Conditional replication and expression system

ABSTRACT

The present invention relates to the utilization of conditionally replicating recombinant nucleic acid molecules rescued from the integrated state for the expression of foreign proteins. The usefulness of the system is illustrated with a conditionally replicating recombinant nucleic acid molecule encoding the adeno-associated virus (AAV) capsid proteins. The present invention also relates to methods employing and conditionally replicating recombinant nucleic acid molecules for the packaging of recombinant AAV nucleic acid molecule into AAV capsids. The present invention also relates to packaging cell lines for recombinant AAV, expressing both the AAV rep and cap-genes.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of molecular biology, inparticular the field of systems for the replication, transcriptionand/or expression (especially translation into protein) of genes orother nucleic acid molecules of interest.

BACKGROUND OF THE INVENTION

So many of these systems have been developed over the last two or threedecades that it is hardly feasible to give a useful summary of the manypossibilities. These possibilities are generally known to the peopleskilled in this art anyway. However, there are a number of genes whichare difficult to replicate, transcribe or express for a variety ofreasons. A quite obvious reason is for instance that the productproduced upon expression is toxic to the cell in which the nucleic acidof interest is expressed. There are however less clear reasons whyreplication, transcription or expression of a nucleic acid of interestdoes not lead to useful levels of replication, transcription and/orexpression. This invention typically deals with the replication,transcription and/or expression of such nucleic acids. The presentinvention was made during research involving adeno associated virus(AAV) and is typically useful for replication, transcription and/orexpression of nucleic acids in an AAV-based system and typically forreplication, transcription and/or expression of AAV-genes, in particularthe cap-gene. However, other genes resisting replication, transcriptionand/or expression in the regular systems or genes or other nucleic acidsthat may only be produced upon induction will also be suitable for usein the presently invented system. The invention will however neexplained in more detail based on the AAV-system. AAV is a virus that istypically suggested for use in gene therapy, whereby a gene of interestis packaged into an AAV virion, which can infect a cell to be providedwith said gene. The present invention arrives at a universal packagingsystem for AAV derived vectors provided with such a gene therapy relatednucleic acid.

AAV is a non-pathogenic human parvovirus (reviewed in^(1, 2)). The virusreplicates as a single strand DNA of approximately 4.6 kb. Both the plusand the minus strand are packaged and infectious. Efficient replicationof AAV requires the co-infection of the cell by a helper virus such asAdenovirus or Herpes Simplex Virus. In the absence of a helper virus, nosubstantial replication of AAV is observed. AAV is therefore alsoclassified as a “Dependovirus”, When no helper virus is present, the AAVgenome can integrate into the host cell genome. The wild-type virus hasa strong preference (70%) for an integration site on the long arm ofchromosome 19 (19 q13.3)³⁻⁵. Following integration, the expression ofthe virus genes is not detectable. The integrated provirus replicates asa normal part of the host cell genome upon division of the transducedcell and ends up in both daughter cells. This stage of the virus lifecycle is known as the latent stage. This latent stage is stable but canbe interrupted by infection of the transduced cell by a helper virus.Following infection of the helpervirus, AAV is excised from the hostcell genome and starts to replicate. During the early phase of thislytic cycle the rep-genes are expressed. Approximately 12 to 16 hourslater, the capsid proteins VP1, VP2 and VP3 are produced and thereplicated virus DNA is packaged into virions (structure of theAAV-genome and its genes is depicted in FIG. 1). The virions accumulatein the nucleus of the cell and are released when the cell lyses as aresult of the accumulation of AAV and the helpervirus (reviewedin^(1, 2)).

The AAV-genome contains two genes named rep and cap (FIG. 1). Threepromoters (P5, P19 and P40) drive the synthesis of mRNAs coding for 4Rep-proteins (Rep78, Rep68, Rep52 and Rep40) and three capsid proteins(VP1, VP2 and VP3). The AAV-genome is flanked on both sides by a 145 bpsequence, called the Inverted Terminal Repeat (ITR), which appears tocontain all the cis-acting sequences required for virus integration,replication and encapsidation^(6, 7).

The capsid proteins VP1, VP2 and VP3 are produced from a 2.6 kbtranscript of the AAV P40 promoter, which is spliced into two 2.3 kbmRNAs by using the same splice donor but two different splice acceptorsites. The splice acceptor sites are located at both sides of the VP1translation start signal. VP1 is translated from the messenger that usesthe splice acceptor directly in front of the VP1 translation initiationcodon. VP2 and VP3 are translated from messengers that are spliced tothe acceptor 3′ of the VP1 ATG. VP2 and VP3 are translated from thismessenger by use of an ACG translation start (VP2) or a downstream ATG(VP3). Since all three coding regions are in frame, the capsid proteinsshare a large domain with an identical amino-acid sequence. VP3 isentirely contained within VP1 and VP2, but the latter two containadditional amino-terminal sequences. Similarly, VP1 contains the entireVP2 protein but carries an additional N-terminal sequence. All threecapsid proteins terminate at the same position⁸. The AAV capsid is 20 to24 nm in diameter^(9, 10) and contains approximately 5% VP1, 5% VP2 and90% VP3. This ratio is believed to reflect the relative abundance of thealternatively spliced messengers and the reduced translation initiationefficiency at the ACG initiation codon for VP2.

During a productive infection, the P5-promoter is activated first anddirects the production of the large Rep-proteins, Rep78 and Rep68. Theseproteins are essential for AAV-replication and trans regulation of viraland cellular genes. The large Rep-proteins activate the P19 and the P40promoter. In a latent infection, however, Rep78 and Rep68 down regulateexpression of the P5 promoter and help to maintain the latency of AAV(for a review see¹). The smaller Rep-proteins, Rep52 and Rep40, areencoded by transcripts from the P19 promoter and are important for theformation of infectious virus¹¹. The P40 promoter is the last promoterto become activated and its activation follows the expression of thelate genes of the helper adenovirus. Via alternative splicing, differentmRNAs are produced coding for the structural proteins VP1, VP2 en VP3¹².

Adeno-Associated Virus Vector Technology

The first recombinant AAV vectors were made by replacing sequences fromthe rep or the cap gene by the sequences of interest¹³⁻¹⁵. Two methodswere used to package the recombinant vector. In one method, the vectorgenome was packaged by co-transfecting into adenovirus infected cells aplasmid containing the vector together with a plasmid containing themissing AAV-gene. In the second method, a plasmid containing the vectorwas co-transfected with an AAV-genome that was too large to be packagedby an insertion of lambda phage DNA¹³⁻¹⁵. Recombinant virus produced inthis way is always contaminated with wild-type AAV (ranging from 10-50%compared to the recombinant titer). This is presumably due torecombination between the two co-transfected plasmids which contain asubstantial region of overlap, or by loss of the lambda DNA sequence.The contaminating wild-type AAV causes a further amplification of therAAV upon infection of a new batch of adenovirus infected cells, leadingto higher rAAV-titers but also leading to amplification of thecontaminating wild-type AAV¹³⁻¹⁵.

To circumvent the production of wild-type AAV, a packaging plasmid wasconstructed that contains no overlap with the vector plasmid⁷. With thispackaging plasmid, it is possible to generate rAAV virus stocks that arefree of detectable amounts of wild-type AAV, while at the same time itenables the production of 0.1 to 1 rAAV particles per cell⁷. Thispackaging system, or analogous systems derived therefrom, are currentlyused by most laboratories. Although this is the method of choice at thismoment, the method is far from optimal since it cannot easily be scaledup to allow industrial production of rAAV vectors. Plasmid transfectionsare inherently inefficient and difficult to standardize or to scale up.This is even more true for co-transfections. In addition, whereas thewild-type virus replicates to 10³-10⁴ particles per cell, the yield ofrAAV in a typical rAAV-production is very low with the current methods(01-1 particles per cell)^(7, 16). This low yield makes purification ofthe rAAV a difficult task to undertake. The production problems pose aserious technological obstacle for the further development of AAV-vectortechnology for, for instance, gene therapy purposes. There is clearly agreat need for an efficient and simple method for the production ofrAAV. A very convenient packaging system would be in vitro packaging ofrAAV by purified recombinantly produced AAV-proteins. A practicalalternative is the generation of a packaging cell line for rAAV wherethe packaging cell line supplies in trans the required AAV and helpervirus proteins for the production of rAAV. The specific recombinant AAVproducing cell lines are then generated by stable transfection of aplasmid containing the recombinant AAV into the packaging cells. Thepresent invention is useful for both the in vitro packaging strategy andthe packaging cell line strategy.

Recombinant AAV Packaging Cell Lines

Packaging cell lines are currently the most efficient way in whichretrovirus and adenovirus vectors are produced for industrial an/ortherapeutic purposes such as gene therapy^(17, 18). Virus proteinproduction for the in vitro packaging on an industrial scale iscurrently employed for Lambda phages, but not for other viruses. Thegeneration of general packaging cell lines for rAAV has been an elusivegoal for many years. Recombinant AAV packaging cell lines require thatthe in trans required AAV-proteins are only functional during theproduction phase of the rAAV vector. Constitutive function or expressionis not desired for at least two reasons: 1) rescue and replication ofthe vector DNA prior to production would interfere with the growth andthe stability of the cell line and 2) specifically the AAV-Rep-proteinsare toxic to cells even in the absence of a recombinant AAV vector. Thelatter is largely due to the well documented, but as yet not explained,anti-proliferative effect of the large Rep proteins^(19, 20). Rep78 andRep68 repress both cellular and viral promoters in transientassays^(21, 22). Upon stable transfection, the large Rep proteinsinhibit cell proliferation^(19, 20). The mechanism is not wellunderstood. It is possible that the observed inhibition of mRNAtranscription and translation represses the production of crucialcellular gene-products^(23, 25). On the other hand, is it possible thatthe large Rep-proteins inhibit DNA replication directly^(26, 22, 27).Considering the pleiotropic effect of Rep-protein expression on cells,it is possible that both effects play a role in the anti-proliferativeeffect of the large Rep-proteins.

Cell Lines With Inducible Rep-Gene Expression

Until now, it has not been possible to make stable cell lines expressingthe large Rep-proteins constitutively (see above). Followingsubstitution of the P5 promoter with an inducible promoter, such as themethallothionine promoter²⁰ or the steroid inducible Mouse Mammary TumorVirus (MMTV) long terminal repeat (LTR) promoter¹⁹, it was possible toisolate out of a large number of clones, one and two clones thatinducibly expressed Rep78, respectively. Rep52 was expressedconstitutively in two of the three clones, whereas Rep68 and Rep40 whichare translated from the spliced mRNAs, were not detectable^(19, 20).Although these clones were able to functionally produce Rep78 and Rep52,the levels were too low for the replication and packaging of recombinantAAV^(19, 20). Apart from this, the percentage of clones expressing Rep78was low. Probably, there was a strong selection against a high level ofrep expression. In case of the MMTV-promoter driven rep-expression, thereplication and production of infectious virus of rep-negativerecombinant AAV constructs could be improved by adding a constructconstitutively expressing Rep40²⁸. Still, at least three problemsremain^(19, 20, 28): 1) The cell lines do not express capsid proteins.Capsid proteins need to be supplied through transfection of a capsidgene construct. 2) Significant replication of rAAV-constructs requirestransfection of the glucocorticoid receptor (in case of theMMTV-promoter). 3) The yield of rAAV is not improved over the transientpackaging systems and thus is not sufficient for industrial use in thesense of production of gene delivery vehicles.

Recently, we were able to generate cell lines with inducible and highlevel rep-gene expression using an improved inducible promoter system.However, also for these cell lines the capsid genes need to be addedexternally during virus production.

Recently, Clark et al. reported the generation of a full complementingcell line²⁹. Although they do not know how to reconcile their resultswith the results of most other laboratories, they succeeded ingenerating cell lines inducibly expressing rep and cap from constructsthat were stably integrated into the host cell genome. Unfortunately,this result was only obtained when the rep-gene, the cap-gene, adominant selectable marker gene and the rAAV-vector sequences werepresent on the same plasmid, thus resulting in dedicated packaging celllines. These packaging cells can, therefore, only be used for theproduction of the specific rAAV introduced together with the rep and capgenes and not be used to produce a different rAAV vector. It was alsoattempted to generate a general packaging cell line²⁹. A cell line wasobtained that inducibly replicated introduced rAAV and expressed thecap-gene. However, the levels of rep-expression where significantlylower than in the dedicated cell lines and although cap-RNA wasproduced, the levels were insufficient to make this cell line suitablefor packaging of recombinant AAV. Since the rep and the cap-genes arephysically linked to each other in this approach, it is not likely thatthe levels of rep and cap can easily be enhanced. For instance, there isthe risk that rep-gene expression in the uninduced state is elevated toa level toxic for the packaging cells. This cell line was intended forand is useful for determining the infectious titer of rAAV preparationsand testing of new rAAV vectors in a transient assay^(29, 30).

SUMMARY OF THE INVENTION

The present invention provides a system for conditionally replicating,transcribing and/or expressing recombinant nucleic acid molecules thatcan be used to obtain very high expression of a foreign gene. Saidsystem is specifically suited for the expression of genes that are notexpressed efficiently by traditional polymerase II promoters, fromnon-replicated DNA or from chromosomally integrated foreign DNA. Inaddition, such a system is suited for the generation of cell linesstably transformed with the gene of interest, which only upon inductionof replication will express significant amounts of product encoded bysaid foreign gene.

Thus, the invention provides a method for conditional replication of anucleic acid of interest which nucleic acid is present, integrated inthe genome of a eukaryotic host cell, comprising providing said nucleicacid with at least one regulatory element which essentially repressesreplication in an integrated setting, but allows replication in anepisomal setting, further providing said nucleic acid with at least ameans for functionally excising said nucleic acid in a functional formupon the presence of a signal, whereby said resulting nucleic acid inepisomal functional form is replicated.

As used herein, essentially repressed means that no replication,transcription or expression of the nucleic acid occurs to anysignificant extent in the sense that it can be used for production ofsaid sequence or a product thereof. At least replication will occur whenthe genome of the host is replicated, but even some additionalreplication, transcription and/or expression may occur.

As used herein, functionally excising means that the sequence ofinterest may be physically excised from the genome, i.e. becomeepisomal, but also that the sequence can be functionally separated fromthe rest of the genome in the sense that it replicates, is transcribedand/or expressed separately from the genome and that the real functionalreplication, transcription and/or expression, only occurs using episomalcopies of the sequence of interest.

As used herein, a signal means any means which is capable of inducingfunctional excision of the sequence of interest. In the case of AAVbased systems, this may be the presence of Rep-proteins, in particularRep68 or Rep78 or a functional fragment or derivative thereof(functional in this case meaning capable of inducing replication of theAAV vector's replication) which induces expression of the cap-gene, orany gene that has been placed under the control of cap-gene regulatoryelements. In the latter embodiment, any gene, or DNA-molecule for thatmatter, can be replicated, transcribed and/or expressed upon inductionby Rep-proteins. In the earlier embodiment, where Cap-proteins areexpressed upon induction, a packaging cell line for AAV based vectors isprovided, thus enabling the production at significant levels of AAVbased gene delivery vehicles, which are thus also a part of the presentinvention, when obtainable by using, for instance, a packaging cell lineaccording to the invention. A gene delivery vehicle is defined herein asan AAV-like virus particle, comprising a nucleic acid molecule based onan AAV-derived vector (having at least a functional AAV-packagingsignal), which nucleic acid molecule comprises a sequence of interest tobe delivered to a cell to be infected by said gene delivery vehicle,especially in the context of gene therapy. A sequence of interest may bea sequence encoding a protein or, for instance, a sequence encoding anantisense RNA or DNA molecule.

It is preferred, according to the present invention, to use a means forfunctionally excising a nucleic acid of interest from the genome whichis based upon a viral replication system. These systems are explained inmore detail herein. One such a preferred system is the presence of twoAAV-ITR's on either side of the nucleic acid or DNA molecule ofinterest. Another preferred system is a system whereby the excisionmeans is derived from a papova virus, preferably a polyoma virus,particularly SV40.

In the first embodiment, the signal for excision is the earlierdiscussed presence of Rep-proteins; in the second embodiment, the signalis the so-called large T-antigen or a functional fragment or derivativethereof. As a secondary level of control, it is preferred to place theexcised sequence under control of an inducible promoter. This leads toclear advantages, for instance, because transcription and translationcan be induced after a time interval of sufficient length to allow for alarge number of copies of the sequence of interest to have beenreplicated. In the production of a packaging system, it is preferredthat the nucleic acid of interest comprises at least a functional partor derivative of a cap-gene of Adeno Associated Virus (AAV). In thiscase, functional means that the protein must be capable of partaking inthe packaging of an AAV-based vector.

The invention further provides a recombinant vector for carrying out amethod, according to the invention, comprising a nucleic acid sequenceof interest, a means for integration into the genome of a eukaryotichost cell, a means for functional excision of the nucleic acid ofinterest after integration upon the presence of a signal, a means foressentially repressing replication of said nucleic acid of interest inthe integrated format and a means for allowing replication of thenucleic acid of interest in the episomal format, whereby one or more ofthe mentioned means may be one and the same. Preferably, such a vectoris one whereby at least one of the mentioned elements is derived fromAAV, or one whereby at least one of the elements is derived from apapovavirus, preferably a polyomavirus, in particular SV40.

The essential elements for a vector derived from AAV are: when apackaging cell line is to be produced, the AAV-ITR's and a functional,cap-gene, whereby functional has been defined hereinbefore.

For a papova based vector an SV40 origin of replication is essential.

Of course, for these vectors, it is again preferred that the sequence ofinterest is placed under the control of an inducible promoter. Theinvention also provides a recombinant eukaryotic host cell comprising avector according the invention, preferably a cell further comprising avector encoding at least a functional part of a rep-gene of AAV. This,of course, is a packaging cell for AAV-based vectors, which, preferably,comprises all AAV genes in trans which are deleted from the recombinantAAV derived vector.

In a further preferred embodiment, the conditionally replicating nucleicacid molecule is stably integrated in the host cell genome, while theDNA encoding a protein or proteins governing the specific replication ofsaid nucleic acid molecule is introduced at the time replication isdesired.

In yet another preferred embodiment of the invention, said nucleic acidmolecule is stably integrated in the host cell genome, while the DNAencoding a protein or proteins governing the specific replication ofsaid nucleic acid molecule is also stably integrated in the host cellgenome. In the latter case, the DNA encoding said protein or proteins isexpressed conditionally. Alternatively, said protein or proteins areengineered in such a way that its replicative functions are in-operativeuntil induced. This can be achieved, for instance, by generatingtemperature sensitive mutants or by generating chimeric proteins fromwhich the activity or compartmentalization in the cell can be regulated.

In another preferred embodiment of the invention, said nucleic acidmolecule comprises the SV40-origin of replication while said protein isSV40 Large T-antigen. As stated hereinbefore, in one particularlypreferred embodiment, said nucleic acid molecule contains two SV40origins of replication, preferably flanking said foreign gene andpositioned in the same orientation on said nucleic acid molecule so asto enable a polymerase chain reaction-like amplification of the internalsequences.

In another preferred embodiment, the invention provides a method for thegeneration of conditionally replicating molecules.

In another embodiment, the invention provides a stable cell lineexpressing AAV-2 capsid proteins VP1, VP2 and VP3 in a regulatedfashion. The relative ratio of VP1, VP2 and VP3 and the absolutequantity of capsid protein in the cell approximate the expression ofcapsid proteins in wtAAV-2—infected cells. The amount of capsid proteinin the cell and the relative ratio of the capsid proteins VP1, VP2 andVP3 enable the production of AAV-capsids and allows for the efficientpackaging of modified wild type and recombinant AAV genomes. A cellline, according to this embodiment of the invention, is useful for thegeneration of a recombinant packaging cell line conditionally expressingboth the AAV-rep gene and the AAV-cap gene. Moreover, said cell line isuseful for the production and purification of AAV-capsid proteins andintact AAV-capsids useful for the in vitro packaging of foreign DNA orincorporation into virosomes.

In yet another earlier discussed embodiment, the invention provides aprocedure for the generation of a fully complementing generalrAAV-packaging cell line. A large variety of recombinant AAV vectors canbe produced by their introduction into said packaging cell line followedby the induction of the lytic cycle. The recombinant AAV DNA can beintroduced by means of transfection or infection into the packagingcells for each production or a stable cell line can be created carryingthe recombinant AAV DNA stabily integrated in the DNA of the packagingcell. In the latter case, production of recombinant AAV can proceed uponinduction of the packaging functions and infection with a helper virussuch as adenovirus. This system offers the advantage that the cell linecan be grown to high cell numbers prior to the production of rAAV, adesirable feature for large scale production purposes. Using theprocedure according to this embodiment of the invention, the productionprocess is simplified significantly due to the reduction of componentsand manipulations that need to be carried out for each production run.It also allows for consistent production since the cell line can beanalyzed and tested for undesirable contaminations and productionfeatures. In addition, it enables the standardization of the productionprocess, since it avoids DNA introduction into cells via transfectionprocedures. The manipulations required are easy to scale up (i.e.induction of rep and cap gene expression by changing the cellsenvironment (i.e. the medium) and the infection with a helpervirus suchas adenovirus). A cell line generated according to the procedure of thepresent invention has the special feature of regulating the AAV-geneexpression such that it reflects the expression of AAV-genes in a normallytic cycle of wtAAV (i.e. early and late expression of rep and lateexpression of cap). It is another aspect of the invention that itprovides a method for the drastic reduction of wtAAV formation duringthe production of recombinant AAV. Although the problem ofwtAAV-formation was reduced by the invention of Samulski et al⁷,recombinant AAV preparations still are often contaminated with wtAAV.Samulski et al removed most of the sequence overlap between thepackaging construct and the recombinant AAV construct leaving only the 6bp overlap of the XbaI site. The formation of wtAAV in this system iseither dependent on homologous recombination events between thepackaging and the recombinant AAV construct using the 6 bp overlap, ordue to non-homologous recombination since there are extremely largeamounts of DNA formed during a productive cycle. The reduction ofoverlap alone necessitates two recombination events between thepackaging and the recombinant AAV construct before wtAAV is formed. Wealso follow a strategy that relies on minimizing the region of overlap.However, the present invention provides a system in which it is possiblefor the first time to separate the rep and the cap-gene constructs. Byphysically separating the transcription units of rep and cap they can beintroduced at different positions in the host cell chromosome withoutloss of 1) expression levels and 2) the regulated and timed expressionof rep and cap in the early and late phase of the lytic cycle. Thephysical separation of the transcription units of rep and cap introducesan extra obstacle for the formation of WtAAV since now threerecombination events are required for the formation of wtAAV. Inaddition, the separation of the transcripts allows for the reduction ofthe amount of AAV DNA in the cell since now only the cap-gene DNAaccumulates to large amounts in the cell. The rep-specific DNA ispresent in a low number of copies in the cell and thus forms aninefficient target for the recombination process.

As used herein, the term “gene” refers to a nucleic acid moleculeencoding a protein and/or RNA.

As used herein, the term “wtAAV” refers to a nucleic acid moleculecontaining the genes rep and cap derived from AAV serotypes 1, 2, 3, 4or 5 or functional analogs or parts thereof physically linked to twoAAV-ITR.

As used herein, the term “recombinant AAV vector” means a nucleic acidmolecule comprising at each end an AAV-ITR. When packaging intoAAV-virions is desired, the size of the recombinant AAV vector islimited by the size constraints for packaging into AAV particles, whichwith the current state of the technology is about 5 kb. The recombinantAAV vector, preferably, does not contain sequences functionallyanalogous to the terminal resolution site in the AAV-ITR, as this mightinterfere with the stability of the recombinant vector. The recombinantAAV vector can contain any sequence, however, preferably, it contains agene with therapeutic properties when introduced into cells of apatient. The gene can be therapeutic directly but, preferably, encodesfor one or more proteins or RNA with therapeutic properties.Non-limiting examples of such genes are the human lysosomalglucocerebrosidase gene (E.C.3.2.1.45); a globin gene from the humanb-globin gene cluster physically linked to sequences from the b-globinlocus control region; a gene encoding an RNA or protein with anti-viralactivity and the human multidrug resistance gene 1 (MDR1).

It will be understood that by the term “recombinant AAV packaging cell”is meant a cell line that provides in trans the required AAV-proteinsnecessary for the replication and/or packaging of modified wild-type orrecombinant AAV genomes. The in trans required proteins are providedeither in a constitutive fashion or in a regulated fashion.

As used herein, the term “functional levels of parvovirus proteinexpression” refers to levels of expression sufficient for replicationand/or packaging of recombinant or modified wild-type parvovirusgenomes.

As used herein, the term “replication”, with respect to viral DNA,refers to a process of multiplication of a nucleic acid moleculedistinct from the normal replication of eukaryotic chromosomal DNA, inthat not just one but, indeed, many copies of the replicating moleculesare formed in a cell during the process.

As used herein, the term “replicating DNA molecule” refers to a DNAmolecule which can undergo replication in a cell. The replication canstart from an integrated DNA molecule or from a DNA molecule that ispresent in the nucleus of a cell as an episome.

It is understood that by the term “regulated expression” is meantexpression levels that can be altered by either manipulating the cellsenvironment, for instance, but not limited to, the addition/removal ofcompounds to/from the medium in which the cells are grown, or bytransfecting into cells DNA, RNA or protein or by infecting the cellswith a virus such as adenovirus or herpes simplex virus.

It will be further understood that by the term “regulated promoter” ismeant a nucleic acid molecule, that enables the regulated expression ofa linked gene.

As used herein, the term “conditionally replicating DNA molecule” refersto a replicating DNA molecule from which the level of replication can bemodified by the function of a protein or proteins of which theexpression can be regulated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure and the genome organization of wtAAV.

FIG. 2 is a schematic representation of the constructs

FIG. 3 depicts regulated expression of Rep78 and Rep68 from ptetrepcapand ptetp5repcap in HtTA cells two days after transfection of theconstruct. Protein extracts from transfected cells were analyzed byimmunoblotting with an anti-Rep antibody 7B7.32, a kind gift from Dr. N.Muzyczka. When indicated (+), the cells had also been infected withadenovirus (multiplicity of infection (moi) 20) at the time oftransfection. When indicated (1), the cells were transfected andcultured in the presence of 1 ug/ml doxycycline. Protein extract fromadenovirus (moi 20) and wtAAV (moi 2) infected HtTA cells (wtAAV) servedas controls for the production of Rep78 and Rep68. Protein from HtTAcells served as a negative control (lane marked with (−))

FIG. 4 depicts a replication center assay specific for recombinant AAVIG-CFT, a recombinant AAV vector containing the human b-globin gene andthe neo-gene³¹. Recombinant AAV was isolated from adenovirus infected(moi 20) HtTA cells transfected with recombinant AAV construct pIG-CFTtogether with either ptetrepcap and ptetp5repcap as packagingconstructs. The packaging plasmid pAAV/Ad served as a positive controlfor the assay and Adenovirus-infected HtTA cells served as a negativecontrol for the assay.

FIG. 5 depicts a regulated expression of Rep78 and Rep68 from ptetrepcapand ptetp5repcap in stably transfected HtTA cells. HtTA cells wereco-transfected with the indicated constructs and pX343, a hygromycin Bresistance gene containing plasmid. The cells were selected forhygromycin B resistance for 14 days starting two days aftertransfection. During transfection and selection, the cells were grown inthe absence (o) or presence (2) of 2 ug/ml tetracycline. Followingselection, the cells were seeded in medium devoid of tetracycline andinfected with adenovirus (moi 20). Protein was extracted after 48 hoursand immunoblotted using an anti-Rep specific antibody 7B7.32. The lanesmarked mock were from left to right, transfected only with thehygromycin construct and untransfected HtTA cells. The lane marked wtAAVcontains protein from HtTA cells infected with adenovirus (moi 20) andwtAAV (moi 2). The lanes marked Rep68 and Rep78 contain purified Rep68and Rep78 respectively, a kind gift from Dr. S. Zolotukhin.

FIG. 6 depicts an expression of pITRp5p40cap in HtTA cells two daysafter transfection. The panel marked Cap, is an immunoblot with ananti-Cap specific antibody B1, a kind gift from Dr. J. Kleinschmidt.Indicated are the locations of the proteins VP1, VP2 and VP3. The panelmarked Rep, is an immunoblot with an anti-Rep specific antibody 7B7.32,a kind gift from Dr. N. Muzyczka. Indicated are the proteins Rep78 andRep68. In the table below is depicted (+) when the cells weretransfected with pITRp5p40cap and/or ptetrep and/or were infected withadenovirus (moi 20) or wtAAV (moi 2) at the time of transfection.

FIG. 7 depicts an expression of VP1, VP2 and VP3 in HtTA cells stabletransfected with pITRp5p40cap (lane 1-8). The cell lines HtTA-cap1(lanes 1,3,5,7) and HtTA-cap2 (lanes 2,4,6,8) were analyzed forcap-expression. Protein was isolated from untreated HtTA-cap cells (lane1 and 2); from cells infected with adenovirus 48 hours before proteinisolation (lanes 3 and 4); from cells transfected with ptetrep 48 hoursbefore protein isolation (lanes 5 and 6) and from cells infected withadenovirus (moi 20) and transfected with ptetrep 48 hours before proteinisolation (lanes 7 and 8). Adenovirus (moi 20) infected HtTA cellsserved as a negative control. The panel marked Cap, is an immunoblotwith an anti-Cap specific antibody B1, a kind gift from Dr. J.Kleinschmidt. Indicated are the location of the proteins VP2 and VP3.The panel marked Rep, is an immunoblot with an anti-Rep specificantibody 7B7.32, a kind gift from Dr. N. Muzyczka. Indicated are theproteins Rep78 and Rep68.

FIG. 8 depicts an expression of cap-specific RNA in HtTA cells stabletransfected with pITRp5p40cap (lane 1-8). Treatment of the cells andlane numbering is the same as in FIG. 7. Lane 10 contains RNA fromuntreated HtTA cells. Instead of protein, total RNA was isolated andNorthern blotted. The filters were hybridized with a cap-specific probethat contained no sequence overlap with ptetrep. Indicated is the 2.3 kbRNA that hybridized to the probe.

FIG. 9. is an expression of VP1, VP2 and VP3 in HtTA-cap2 cells (lane1-4). Shown is an immunoblot with an anti-Cap specific antibody B1 ofprotein isolated from untreated cells (lane 1), from cells that had beeninfected with adenovirus (moi 20) two days before protein isolation(lane 2), from cells transfected with ptetrep two days before proteinisolation (lane 3) and from cells infected with adenovirus (moi 20) andtransfected with ptetrep two days before protein isolation (lane 4). Thelane marked HtTA wtAAV contains protein from HtTA cells infected withadenovirus (moi 20) and wtAAV (moi 2) two days before protein isolated.VP1, VP2 and VP3 are indicated.

FIG. 10 depicts a Hirt-extract DNA from HtTA-cap1 cells and HtTA-cap2cells. Treatment of the cells and lane numbering is the same as in FIG.7. Lane 10 contains Hirt-extract from untreated HtTA cells. Episomal DNAwas isolated and analyzed on a Southern blot. The filters werehybridized with a cap-specific probe that contained no sequence overlapwith ptetrep. Indicated are the duplex monomer (dm), the duplex dimer(dd) and an adenovirus cross hybridizing band (Ad).

FIG. 11 depicts a Hirt-extract from HtTA cells infected with rAAVproduced by HtTA-cap2 cells. Shown is a Southern blot hybridized with acap-specific probe that contains no sequence overlap with ptetrep. Oneday before Hirt-extraction, the HtTA cells were infected with rAAV andadenovirus (lane 1, 2) and transfected with ptetrep (lane 1). As anegative control, a mock rAAV-isolate from HtTA-cap2 cells infected withonly adenovirus (moi 20) was used to infect HtTA cells also infectedwith adenovirus (lane 3).

FIG. 12 depicts a Southern blot of Hirt-extract from adenovirus HtTAcells infected with rAAV produced by HtTA-cap2 cells. The Southern blotwas hybridized with a cap-specific probe that contains no sequenceoverlap with ptetrep. One day before Hirt-extraction, the adenovirusinfected HtTA cells were transfected with ptetrep (lane 1 and 3) andeither infected with rAAV (lane 1 and 2) or infected with rAAV that hadbeen pretreated with DNase I (1 mg/ml) for 30 min. at 37° C. andchloroform (lane 3 and 4).

FIG. 13 depicts a Rep-gene expression two days after transfection ofplasmid ptetrep (lane 1), ptetp5repcap (lane 2), ptetp5repEcoNI (lane 3)and ptet*p5repEcoNI (lane 4) into adenovirus infected HtTA cells. Shownis an immunoblot with an anti-rep antibody 303.9, a kind gift of Dr. J.Kleinschmidt. Indicated are the locations of Rep78, Rep68, Rep52 andRep40.

FIG. 14 depicts a Hirt-extract DNA from HtTA cells infected with rAAVproduced by HtTA-cap2 cells. Adenovirus infected HtTA cells weretransfected with ptetrep and infected with rAAV produced on HtTA-cap2cells transfected with ptet*p5repEcoNI (lane 1), ptetp5repEcoNI (lane2), ptetp5repcap (lane 3) or ptetrep (lane 4). Hirt-extract was isolated24 hours after transfection and infection. The extract was digested withDpnI, Southern blotted and hybridized with a cap-specific probe thatcontains no sequence overlap with ptetrep. Indicated are the duplexmonomer and the duplex dimer of the cap-gene containing rAAV rescued,replicated and packaged in the transfected HtTA-cap2 cells.

FIG. 15 depicts an expression of Rep proteins in transfected andHygromycin B selected HtTA-cells. HtTA cells were transfected in thepresence of doxycycline with a mixture of pTK-Hyg (Clontech, Palo Alto)and rep-expression plasmids ptetrep (lane 2) or ptetp5repEcoNI (lane 3)Between 30 (ptetrep) and 90 (ptetp5repEcoNI) Hygromycin B resistantcolonies were picked and split in two dishes. The cells from one dishwere washed to remove the doxycycline and infected with adenovirus.Protein was isolated after 48 hours and equal amounts of protein fromsix independent colonies were pooled and analyzed for the expression ofrep-proteins by Western blot. Rep-proteins were visualized with theantibody 303.9. The positions of Rep78, Rep68, Rep52 and Rep40areindicated. Protein from ptetp5repcap transfected and adenovirus infectedHtTA cells served as a positive control (lane 1). A cross-reactinga-specific band is detected with this batch of antibody (*)

FIG. 16 depicts a Western dot blot assay. HtTA cells were transfected inthe presence of doxycycline with a mixture of pTK-Hyg (Clontech, PaloAlto) and rep-expression plasmids ptet*p5repEcoNI or plasmidptetp5repcap. Colonies from only pTK-Hyg transfected HtTA cells servedas a negative control. Hygromycin B resistant clones were picked andcells from each clone were divided over two dishes. One dish was used tocontinue culture of cells for future reference while the cells from theother dish were used to analyze Rep-protein expression. To induceRep-expression, the cells were washed to remove the doxycycline andinfected with adenovirus. Protein was isolated after 48 hours and 30 or3 microgram of protein was spotted on nitrocellulose filters.Rep-proteins were visualized with antibody 303.9. Protein from 52colonies from ptet*p5repEcoNI/pTK-Hyg transfected HtTA cells (A1-A12;B1-B12; C1-C12; D1-D12 and E1-E4). Protein from 24 colonies fromptetp5repcap/pTK-Hyg transfected HTTA cells (E6-E12, F1-F12, G1-G5).Protein from 5 colonies from pTK-Hyg transfected HtTA cells (E5, G6-G9).At location G10, protein from a transient transfection ofptet*p5repEcoNI in adenovirus infected HtTA-cells was spotted. Arrowsindicate protein samples of clones reacting with the antibody 303.9.

FIG. 17 shows a Rep-expression in HtTA cells stabily transfected withptetp5repcap or ptetp5repEcoNI. HtTA-rep1 (lane 2) and HtTA-rep2 (lane3) cells were washed to remove the doxycycline and infected withadenovirus. Protein was harvested after 48 hours and Western blotted.Rep-proteins were visualized with the antibody 303.9. Protein fromadenovirus infected HtTA cells served as a negative control (lane 1). Across-reacting a-specific band is detected with this batch of antibody(*).

FIG. 18 shows the rescue and replication of rAAV in HtTA-rep1 (lane 2)and HtTA-rep2 (lane 3) cells. HtTA-rep1 and HtTA-rep2 cells were washedto remove the doxycycline, infected with adenovirus and transfected withrAAV plasmid pTR-Luc. After 36 hours, extrachromosomal DNA was isolatedby Hirt-extraction and digested with DpnI. The DNA was electrophoresedand Southern blotted. Recombinant AAV specific DNA was visualized with aLuc specific probe. Indicated are the duplex monomer and the duplexdimer replication products. Hirt-extract DNA from adenovirus infectedHtTA cells served as a negative control (lane 1).

FIG. 19 shows the rescue and replication of pITR6.5cap in adenovirusinfected CARE.1 cells. CARE.1 cells were seeded in medium withoutdoxycyline and either infected (lane 7) or not infected (lane 6) withadenovirus. After 36 hours, extrachromosomal DNA was isolated, run onagarose and Southern blotted. In panel A is depicted a 10 minuteexposure of a Southern blot hybridized with a cap-specific probe.Puromycin resistant HtTA-rep1 cells derived from clone B1 infected (lane5) or not infected (lane 4) served as negative controls. In lane 1 isloaded Hirt-extract DNA of a transient transfection of pITR6.5cap andptet*p5repEcoNI into adenovirus infected HtTA cells. Lane 2 and lane 3contain Hirt-extract DNA from adenovirus infected HtTA cells andadenovirus infected HtTA-rep1 cells, respectively.

Panel B shows an overnight exposure of the same DNA as in panel A, butit is now hybridized with a rep-specific probe.

FIG. 20 shows an AAV-protein expression in CARE.1 cells CARE.1 cellswere seeded in medium without doxycyline and either infected (lane 6) ornot infected (lane 5) with adenovirus. After 48 hours, protein wasextracted and Western blotted. Rep-proteins were visualized withantibody 303.9. A cross-reacting a-specific band is detected with thisbatch of antibody (*). Capsid proteins were visualized with antibody B1.Lane 1 shows protein harvested 48 hours after transfection of atransient transfection of pITR6.5cap and ptet*p5repEcoNI into adenovirusinfected HtTA cells. Lane 2 shows protein from adenovirus infected HtTAcells. Lane 3 and lane 4 show protein from uninfected and adenovirusinfected puromycin resistant HtTA-rep1 cells derived from clone B1,respectively.

FIG. 21 shows regions of homology in the genetic elements important forthe production of rAAV in the rAAV-packaging cell line of the presentinvention. Depicted are a rep-expression cassette, an excisable andreplicateable cap-expression cassette and a rAAV molecule. The region ofhomology or overlap between the different constructs is highlighted withgrey boxes. Possible homologous recombination sites are indicated with across (x). TR=AAV inverted terminal repeat. ADA cDNA is part of thehuman Adenosine Deaminase cDNA. ADA intron 2 is part of human AdenosineDeaminase intron 2.Chrom. DNA is a schematic representation ofchromosomal DNA flanking the rep-expression cassette. Tet(o) indicatesthe tet^(R)-VP16 binding region in the promoter driving rep-expression.P5, p19 and p40 depict the position of the endogenous AAV-promoters p5,p19 and p40.

DETAILED EXPLANATION OF THE INVENTION ON THE BASIS OF AAV

The present invention is based on our finding that cap-gene expressionis absent or just barely detectable when a cap-gene, physically linkedto a variety of promoters, is introduced stabily, into cells. Similarresults were obtained using cap-constructs in which a cap-gene wasplaced under transcriptional control of its native promoter or of aninducible promoter to avoid possible toxicity. These results werestriking, since the same promoters directed moderate to very highamounts of cap-gene expression when introduced transiently into cells.In the latter case, most of the introduced DNA is present in an episomalform and not integrated into the host cell genome, whereas stablytransduced cells carry integrated constructs. Moreover, compared to thenumber of copies of the transgene in stably transfected cells, thenumber of copies of the transgene in transiently transfected cells ismuch higher. According to the present invention, cap-expression isdependent on the presence of the capsid DNA in an episomal form in thenucleus of cells, preferably in high copy numbers. This also explainsearlier observations that in a lytic infection of AAV, first therep-gene is expressed, and only later in the infection, when sufficientmolecules have accumulated in the nucleus of the cells, the cap gene isexpressed in significant quantities. The switch from an early geneexpression pattern to a late gene expression pattern for AAV isdetermined by the number of cap-gene copies that have accumulated due tothe early gene expression of AAV. A regulating mechanism based on thenumber of accumulated gene copies is a striking adaptation in anevolutionary perspective. AAV is a dependovirus and, as such, relies forits replication on a co-infecting helper virus. However, it alsocompetes with the co-infecting virus for host cell derived proteins andbasal components, such as nucleotides and energy-carriers. One way to besuccessful under this pressure is to ensure maximal replication in theearly phase of the replication cycle. It is not desirable to efficientlypackage genomes and thereby to extract templates from the replicationpool early in the replication cycle, since it significantly slows downthe otherwise exponential increase in the number of templates. When thenumber of gene copies is low, efficient packaging function is notdesired since it removes AAV templates from the replication pool, whichinterferes with the accumulation of maximal amounts of AAV-DNA in theshortest possible time. For autonomous viruses, early (replicative) andlate (packaging) gene expression patterns are also favored to circumventand out-compete the immune apparatus. However, for such viruses a strongdependency on copy numbers is probably not so crucial as in aco-infection setting.

The underlying trigger by which an increase in the number of copies isdetected and stimulates the accumulation of cap-RNA is not known. Onepossibility is that negative acting proteins, such as repressors, bindto the AAV-cap DNA and prevent transcription. An increase in the numberof templates also increases the number of binding sites for therepressor. When replication continues, a point is reached where there isnot sufficient unbound repressor left in the nucleus of the infectedcell to inhibit all new templates formed. When replication continuesbeyond this point, templates are no longer bound by the repressor andtranscription can proceed.

In the present invention, a conditionally replicating system triggersthe expression of an otherwise difficult to express gene, such as theAAV-cap-gene. One system relies on the AAV-replication machinery.However, to avoid use of AAV-ITR sequences for the rescue, replicationand accumulation of the cap-gene, alternative replication systems can beused. The perhaps most well known replication system in the art is thereplication system based on the Polyoma virus simian virus 40 (SV40).The Polyoma viruses (Polyomavirinae) are a subfamily of thePapovaviridae, a family of small, non-enveloped viruses with icosahedralcapsids. Members of this family are, among others, the rabbit papillomavirus, mouse polyoma virus and simian virus 40 (SV40). Their genomes aresingle molecules of covalently closed, superhelical, double stranded DNAthat replicate in the nucleus (reviewed in³²) These small viruses,particularly SV40, have been intensively studied. Their genomes can bedivided into early and late regions. In addition to this, each Polyomavirus genome contains a single unique origin of DNA replication. Theearly region is transcribed and expressed early in the replicative cycleand continues to be expressed at late times after infection. In case ofSV40, the early region encodes the small and large T-antigen. The largeT-antigen is the only viral protein required for viral DNA replication.Large T-antigen will also replicate heterologous DNA moleculescontaining an SV40 origin of replication. This property of SV40 largeT-antigen was exploited in COS-cells. The COS-1 and COS-7 monkey kidneycell lines express wild-type SV40 large T-antigen and contain integratedSV40 DNA carrying a deletion of sequences within the origin ofreplication of SV40³³. When SV40 origin of replication containingmolecules are introduced into COS cells, the COS cells will replicatethese heterologous molecules.

The present invention uses the SV40 replication system for theintracellular amplification of cap-DNA. The subsequent enhancedcap-expression can thus be used to package rAAV.

AAV-ITR sequences useful in the invention are obtained from AAVserotypes 1, 2, 3, 4 or 5. Alternatively, mutant or recombinant ITRsequences can be used, which retain the essential properties of theAAV-ITR prototype, which are described in Lefebyre et al³⁴.

For most applications of the invention, the helper virus functions arerequired for efficient packaging of recombinant AAV. In these cases, thehelper virus is inactivated or separated physically from the recombinantAAV virions before using the recombinant AAV virions for thetransduction of cells. The present invention, however, also provides amethod to package recombinant AAV vectors by adding the recombinant AAVDNA to protein extracts or mixtures of protein extracts of cells thatexpressed all or part of the relevant proteins for the replication andpackaging of recombinant AAV. When protein extracts are used from cellsexpressing only some of the relevant proteins for packaging ofrecombinant AAV, the missing proteins can be supplied externally inpurified form. Thus, the present invention obviates the need for usinghelper virus in the production process.

The rep-gene can be derived from anyone of AAV serotypes 1-5, orfunctional analogs or parts thereof, including but not restricted tothose obtained through the introduction of non-essential mutations inthe rep-genes or through the isolation of genes with similarcapabilities, such as the Human Herpesvirus 6 AAV-2 rep genehomologue^(35.)

The cap-gene can be derived from any one of the AAV serotypes 1-5 orfunctional analogs or parts thereof, obtained, but not restricted to,through non-essential mutations in the cap-genes. In addition, thecap-gene sequences can be altered through the replacement or addition ofsequences rendering the produced virion with new or altered target cellspecificity or with improved features for the purification andconcentration of rAAV particles, such as a peptide tag.

Recombinant AAV virions produced according to the invention are purifiedand concentrated using biological, physical or chemical separationtechniques known in the art including, but not limited to, antibodyaffinity purification, density gradient centrifugation or ion exchangechromatography. Alternatively, said virions can be used in a crudeunpurified form.

As used herein, the term “functional analog or part” refers to aderivative with the same activity in kind, though not necessarily in thesame amount, as the original.

It is to be understood that only certain embodiments of the inventionare illustrated by examples and that the examples should not beconsidered restrictive in character. All modifications within the scopeof the invention that may be contemplated by persons skilled in the artare considered to be part of the present invention.

The invention is illustrated by the following non-limiting exampleswherein the following materials and methods were employed. The entiredisclosures of each of the literature references cited hereinafter areincorporated by reference herein.

One of the embodiments of the invention illustrated by the example isthe generation of a rep and cap complementing packaging cell line forthe production of rAAV. Examples 1 and 2 describe the generation of astable cell line expressing the rep-gene. Examples 3 and 4 describe thegeneration of a cell line expressing the cap-gene. For both genes, asolution to the expression problem was found in inducible expressionsystems developed for this purpose. For the rep-gene products, animproved tetracycline regulated expression system was developed. For thecap-gene products, the invention teaches that high level expression isonly obtainable upon replication and accumulation of the cap-DNA. Aconditionally replicating system was developed in which cap-expressioncan be induced. An added advantage of the conditionally replicatingsystem is that expression of cap can be regulated such that expressionoccurs late in the replication cycle of AAV, thus mimicking the wildtype AAV expression pattern in a lytic infection. Example 5 describes analternative method to obtain a conditionally replicating system for theexpression of cap-gene products. Example 6 describes methods for thegeneration of a rep-and cap-complementing cell line useful for thegeneration of recombinant AAV.

EXAMPLES Example 1

Transient Expression of Rep and Cap From Tetracycline Repressor OperatorRegulated Constructs

The classical inducible eukaryotic promoters like, for instance, heatshock promoters, the mouse methallothionine promoter or the mousemammary-tumor virus long terminal repeat promoter, respond to heatshock, heavy metals or hormones³⁶⁻⁴³. These promoters are, in principle,not ideally suited to inducibly express (toxic) genes, since thepromoters are not completely silent in the uninduced state³⁹ and/orbecause the inducing principle induces pleiotropic effects in the targetcells⁴⁴. A new generation of regulated promoters has adapted parts frombacterial transcription units for use in eukaryotic cells⁴⁵⁻⁵². Theseartificial transcription units are adapted from the lacrepressor-operator-inductor system or the TN10-specific tetracyclineresistance operon from E. coli. Three different systems have beendescribed: i) the prevention of transcription initiation by well placedrepressor-operator complexes on the promoter^(45-47, 51); or ii) byblocking the RNA-polymerase II during elongation by a repressor-operatorcomplex^(48, 51); or iii) the activation of a minimal TATA-boxreplenished with operator sequences which can be recognized by anartificial transactivator (tA). The tA consists of the operator bindingcomponent derived from the lac-repressor or the tetracycline repressorand the transcription activating domain from VP16, a Herpes SimplexVirus encoded transcription activator^(49, 50, 52).

Specifically, the latter system, where a minimal promoter is combinedwith operator sequences from the tet-repressor, is suitable for theregulated expression of foreign genes in eukaryotic cells. Since thetet-repressor operator sequences have no functional homologue ineukaryotic cells, these promoters are practically inactive in thepresence of low concentrations of tetracycline or related compounds suchas doxycycline^(52, 53). Stable cell lines exist that constitutivelyexpress the tetracycline-repressor VP16 fusion gene (tA). We have used aHeLa cell line HtTA expressing the tA constitutively⁵² to test thepossibility of obtaining tetracycline regulated expression of rep.

For this purpose, we generated constructs containing the rep and capcoding regions, in which large Rep protein expression was placed undercontrol of the tet-operon (FIG. 2). In the construct ptetrepcap, weexchanged the p5-promoter with the tet(o) promoter, whereas in theconstruct, ptetp5repcap, we cloned the tet(o) in front of the p5promoter. It has previously been shown that the adeno-associated virusP5 promoter contains two motifs centered at −60 and +1 relative to itstranscription initiation site that mediate transactivation by the 13SE1A protein. A cellular factor, YY1, that binds to the motif wasidentified, and its cDNA was cloned. YY1 is a 414-amino-acid zinc fingerprotein that represses transcription when bound upstream of heterologousbasal promoters; E1A-proteins relieve the repression and activatetranscription through YY1⁵⁴. With the ptetp5repcap construct we intendedto study whether additional silencing of the tet-operon was beneficialfor the generation of inducible rep-expressing cells. To determine theactivities of the promoters, the constructs were transfected intoHtTA-cells in the presence or absence of doxycycline and in the absenceor presence of adenovirus. Protein was extracted after two days andWestern blotted. The filters were incubated with the Rep78, Rep68specific monoclonal antibody 7B7.32 (a kind gift of Dr. R. J. Samulski).Both ptetrepcap and ptetp5repcap expressed Rep78 and Rep68 in adoxycycline regulated fashion in HtTA cells and, for both constructs,the expression was upregulated upon adenovirus infection (FIG. 3).Expression of Rep78 and Rep68 in the uninduced state (i.e. withdoxycycline and without adenovirus) is markedly reduced with theptetp5repcap, as compared to ptetrepcap under the same conditions. Theseresults prove that i) it is possible to obtain regulated expression ofRep78/Rep68 using the artificial tet-operon and ii) adenovirus infectionup-regulates expression of the rep-gene in both constructs. In thetetp5repcap construct, the adenovirus effect could be mediated byadenovirus responsive elements in the p5 promoter, such as the twoYY1-sites. In the tetrepcap construct, however, the expression enhancingeffect of adenovirus infection must be mediated by an alternatemechanism, possibly by a direct effect on the tet(o) and the minimalTATA-Box or an adenovirus responsive element in the AAV-genome or bypost transcriptional mechanisms. We observed that the basalrep-expression of the ptetp5repcap construct is reduced compared toptetrepcap. The difference between the constructs is a smallAAV-fragment containing part of the p5-promoter and the 5′ untranslatedregion mRNA encoding the large Rep-proteins. Identified cis-actingsequences in the extra fragment that alone or in combination couldmediate the reduced basal level expression are a major latetranscription factor (MTLF) site, the two YY1-sites⁵⁴ and theRep-binding site⁵⁵ in the P5 promoter.

To verify whether the rep-gene expression was functional, the constructswere used to produce recombinant AAV. The constructs were co-transfectedwith a plasmid pIG-CFT, carrying a recombinant AAV vector³¹, intoadenovirus infected HtTA cells. After three days, recombinant AAV wasisolated and titrated in a replication center assay (RCA) by serialdilution of the recombinant AAV stock on adenovirus and wtAAV infected293 cells⁵⁶ as described in Einerhand et al³¹. The ptetrepcap packagingconstruct produced recombinant IG-CFT with a titer of 20×3×1000=6×10⁴Infectious Particles (IP) per ml, whereas the ptetp5repcap packaging*construct produced recombinant IG-CFT with a titer of 6×3×1000=1.8×10⁴IP per ml (FIG. 4). Thus the AAV-protein coding domain is functional inboth constructs

Example 2

Stable Cell Lines Expressing High Regulated Levels of Rep78 and Rep68

We next generated stable cell lines expressing in a regulated fashionhigh levels of Rep78 and Rep68. HtTA cells were transfected withptetrepcap or ptetp5repcap together with plasmid pX343 containing ahygromycin B resistance gene under transcriptional control of the SV40promoter in a ratio of 10:1 (w/w), respectively. Transfections wereperformed in the presence or absence of 2 ug/ml tetracycline. Two daysafter transfection, the medium was replaced by medium containing 400ug/ml Hygromycine B with or without 2 ug/ml tetracycline. This mediumwas refreshed every 3 to 4 days. After two weeks, the colonies in thedishes were trypsinised, seeded in medium without tetracycline andinfected with adenovirus. After two days, the cells were collected andtotal protein was Western blotted. Rep-protein expression was analyzedusing the Rep78/Rep68 specific monoclonal antibody 7B7.32 (FIG. 5). Inuntreated cells (lane HtTA) and in pBluescript transfected HtTA cells(pBlue), no Rep-specific bands are detected. In the positive controllane, HtTA infected with wtAAV-2, Rep78 and Rep6B are easily detected.Rep68 runs as a double band in SDS-page (M.E. unpublished results).Purified Rep78 and Rep68 protein (kindly provided by Dr. Nick Muzyczka)are in the lanes marked as Rep78; Rep68 served as size markers for therespective proteins. When pAAV/Ad⁷ was transfected or when theptetrepcap construct was transfected, no Rep78 or Rep68 could bedetected in the adenovirus infected pools irrespective of whether thepools were generated in the absence or presence of tetracycline (FIG.5). This complies with the notion that even low levels of Rep78 andRep68 are not tolerated by the cells. The ptetp5repcap construct, incontrast, does express significant amounts of Rep78 and Rep68 in aregulated fashion (FIG. 5). Pools generated in the presence oftetracycline, when the tet(o) is inactive, express Rep78 and Rep68 uponremoval of the tetracycline from the medium. However, when pools weregenerated in the absence of tetracycline, when the tet(o) is active, noexpression of Rep78 and Rep68 could be detected. These results indicatethe presence of sequences in the p5 promoter that down-regulateexpression of Rep78 and Rep68. The sequences in the AAV p5-promoterresponsible for this phenomenon could be the binding sites foradenovirus major late transcription factor (MTLF), Rep78 and Rep68 orYY1⁵⁴.

Example 3

Low Cap-Gene Expression in Stable Transfected Cell Lines

We analyzed the expression of the cap-gene in the stable transfectedpools described above. No expression of VP1, VP2 or VP3 could bedetected (not shown). The presence or absence of tetracycline and/oradenovirus had no effect on expression of cap. Assuming that the p40promoter was not functional in the context of the constructs we hadgenerated, we designed three new constructs. In these constructs,different promoters, including inducible promoters, were cloned in frontof the cap-gene. The constructs ptetcap, ptetp4ocap, pMLPicap (FIG. 2)were transfected in HtTA cells in the presence or absence of adenovirusand, for the tet(o) containing constructs, also in the absence andpresence of doxycycline. After two days, the cells were harvested andprotein was analyzed on Western blot for cap-protein expression. Allconstructs were able to express cap, albeit not under all conditionstested (not shown). Adenovirus infection had a general enhancing effecton expression of cap and for the tet(o)—containing constructs,expression of cap increased upon induction of tet(o) (not shown). Wenext generated stable cap cell lines by transfecting the cap-constructsin HtTA cells. For tet(o) containing constructs transfections wereperformed both in the presence and in the absence of tetracycline.Expression of the cap-gene was analyzed two days after the cells wereseeded in medium without tetracycline and were infected with Ad.Cap-encoded protein could not be detected in these cell lines (notshown). We concluded that the various promoters were not capable ofdirecting cap-expression in stable transfected cells, an unexpectedresult since the same promoters were capable of directing cap-expressionwhen transiently transfected.

Example 4

High Level Cap-Gene Expression in Stable Transfected Cells

The finding that stable transfectants did not express significantamounts of cap-protein was unexpected. Several reasons can be putforward to explain this result. For instance

i) cap-expression is toxic to cells. It is not very likely thatcap-expression is toxic to the cells since there was no difference inthe number of colonies with control transfections and we observed noincrease in the number of hygromycin B resistant colonies with induciblecap-constructs (not shown).

ii) cap-gene expression is inhibited once the cap-gene is integratedinto the host cell genome.

iii) cap-DNA needs to be present in the cell in many copies to titrateout a putative cap-DNA binding factor that inhibits expression from aproximal promoter.

iv) cap is low for any of the promoters tested, whether the construct isintegrated in the host cell genome or not. Expression would then becomedetectable when sufficient templates for transcription are present inthe cells, as in a transient CaPO4 transfection.

To test whether one or more of these hypotheses were valid, we generateda construct that, when integrated in the host cell genome, could beinduced to excise and replicate. For this purpose, we generated theconstruct pITRp5p40cap, in which the cap-gene is flanked by two AAV-ITR.In cells stably transfected with this construct, the cap-gene can beexcised from the genome and replicated by supplying the Rep-proteins intrans. The replicated cap-gene copies accumulate extrachromosomaly, thusmimicking the extrachromosomal state of the DNA in a transienttransfection. One of the differences with a transfection of a plasmid isthe linear state of the replicated DNA. We chose a replicating systemfor the reason that in a productive wild-type AAV infection, thetemplate for cap-expression is also replicated.

We first tested the transient expression capabilities of the constructin HtTA cells (FIG. 6). No expression was detectable when the constructwas transfected alone or together with ptetrep. When the construct wastransfected into adenovirus infected HtTA cells VP3, protein wasexpressed (FIG. 6, lanes 3, 4). However, when the construct wastransfected into adenovirus infected HtTA cells together with ptetrep,all three capsid proteins were readily detectable.

Next, we examined the stable expression capabilities of the pITRp5p40capconstruct. The construct was transfected into HtTA cells together withthe plasmid pX343 carrying the hygromycin resistance gene in a ratio of(10 to 1). The cells were selected after 14 days for hygromycin Bresistance and two independent polyclonal cell lines, designatedHtTA-cap1 and HtTA-cap2, were generated. No expression of cap-proteinscould be detected (FIG. 7, lanes 1-2). Expression of adenovirus proteinsor rep-proteins did not transactivate the p40-promoter (FIG. 7, lanes3-4 and 5-6 respectively). The latter observation is in accordance withprevious art in which transactivation of the p40-promoter by the largeRep-proteins was found in some cases and not in others^(21, 57).However, when HtTA-cap1 and HtTA-cap2 cells were both infected withadenovirus and transfected with ptetrep two days before harvest, VP3protein was readily detectable. VP1 and VP2 were also detected but thesignal was not high (FIG. 7, lane 7-8). Translational mechanisms for thepresence or absence of cap-proteins could be excluded since proteinexpression correlated with the presence of cap-specific RNA (FIG. 8). Toverify that all three capsid proteins could be produced, we repeated theexperiment on HtTA-cap2 cells. This experiment confirmed the previousexperiment but now VP1, VP2 and VP3 are clearly distinguished (FIG. 9).The relative abundance of the three proteins resembles that of awild-type AAV infection. This is important to warrant the production offunctional particles. For instance, similar to the result mentionedabove, Dr. J A Kleinschmidt observed in stable cell lines a lowexpression of cap protein. However, in addition to the low level ofexpression, VP2, which is crucial for capsid formation⁵⁸, wasconsistently not detectable (personal communication).

To test the presumption that cap-DNA was excised from the genome andreplicated, Hirt-extract DNA was analyzed. Expression of cap-proteincorrelated with the detection of recombinant AAV replicationintermediates in the cells (FIG. 10). Thus, one or more steps involvingrescue from the genome, and subsequent replication and accumulation ofthe capsid gene, were required before cap-expression could be detected.

To verify whether the detected cap-expression led to the production offunctional and intact capsids, HtTA-cap2 cells were infected withadenovirus and transfected with ptetrep. After 48 hours, a crude rAAVwas prepared. The crude rAAV preparation was used to infect normal HtTAcells infected with adenovirus and transfected with ptetrep. After 24hours, Hirt-extract DNA was Southern blotted and hybridized with acap-specific probe. Replication of the cap-containing rAAV was readilydetectable (FIG. 11, lane 1). When a control rAAV-preparation wasisolated from HtTA-cap2 cells infected with adenovirus in the absence ofa rep-construct (i.e. when cap-expression was not detected), noreplication could be detected upon infection of permissive HtTA cells(i.e. no transmissible rAAV had been produced). DNaseI digestion andchloroform treatment reduced the concentration of transmissible rAAVapproximately two-fold FIG. 12, indicating the formation of intactAAV-particles by the HtTA-cap2 cell line.

Example 5

Stable Cell Lines Expressing Cap in a Regulated Fashion Using LargeT-Antigen Based Replication Systems

An SV40 replication system useful for the expression of cap may beprepared as follows. Two SV40 origins of replication are cloned as adirect repeat flanking the cap-gene, allowing a PCR like amplificationof the cap-DNA in permissive cells. Expression of cap can be regulatedby the SV40 early gene promoter, the endogenous p40 promoter or otherstrong constitutive promoters. The construct can be co-transfected intocells together with pX343 or other plasmids containing dominantselectable marker genes according to methods described above. Cell linesthat can be used for the generation of the rAAV-packaging system shouldmeet the following criteria: the cells should be able to sustainAAV-replication and SV40 replication; the cells should be compatiblewith the tetracycline regulated expression system described by Bujard etal (i.e. they should not exhibit a high basal level expression of thetet(o) minimal TATA-box element); the cells should not express E1A sinceit would activate the E1A responsive element in the p5-promoter fragmentused in the improved tet(o) described in example 1; the cells should bepermissive for YY1 mediated silencing of the p5-promoter; and, finally,the cells should, preferably, not exhibit rep-mediated inhibition ofSV40 replication, like, for instance, COS cells⁵⁹. A non-limitingexample of such a cell line is COS-1⁵⁹.

Upon co-transfection of the construct with pX343, hygromycin B resistantcolonies can be generated by adding the appropriate concentration ofhygromycin B to the medium of the transfected cells. Individual coloniescan be expanded and analyzed for large T-antigen induced replication andexpression of cap by analyzing protein and Hirt-extract DNA from thecells one or two days after a transfection of the cells with anexpression construct of large T-antigen.

Example 6

Generation of a General Packaging Cell Line For rAAV-Vectors

A cell line based on tetracycline and adenovirus regulated expression ofrep, and AAV-ITR based replication and expression of cap, can begenerated using the following general protocol. Transfection can beperformed of tetp5repcap or suitable derivative with deletions in cap,into HtTA-cap2, together with a plasmid containing the dominantselectable marker gene gpt or zeocin (Stratagene) under transcriptionalcontrol of an SV40 early promoter. Transfection and subsequent selectionshould be performed in the presence of tetracycline or suitable analogsthereof. Deletion of cap-sequences from the rep-gene expressionconstruct is desired to minimize the regions of overlap between thisconstruct and the cap-expression construct, since extensive overlap willincrease the chance of homologous recombination and facilitates theformation of wtAAV. However, while deleting cap-sequences from therep-construct, it is important to leave enough information as to warrantadequate splicing of the messenger to produce Rep68 and Rep40.Rep-constructs with only the rep-coding domains, such as ptetrep, do notsplice efficiently enough to produce significant amounts of Rep68.Splicing information can be contained in cis-acting sequences 3′ of therep-coding region in the form of splicing enhancers or inefficientsplicing of rep-RNA in ptetrep can be the consequence of an interruptionof an, as yet, unidentified mRNA producing a novel AAV protein(Kleinschmidt, personal communication). Following the appropriateselection for expression of the selectable marker gene, differentcolonies can be analyzed for inducible expression of rep by removing thedoxycycline from the medium and infecting the cells with adenovirus.Separate colonies can be screened for the production of cap-DNAcontaining rAAV particles as described above. These cell lines could beuseful for the production of different rAAV by transfecting the DNAcontaining the rAAV into the cells. A stable cell line can be generatedor the DNA can be introduced by transfections for each rAAV preparation.A disadvantage of the packaging cell line is that such rAAV preparationswill also contain a significant amount of cap-DNA containing rAAV. Thiscan be reduced by altering the pITRp5p40cap construct in such a way thatpackaging of the replicated DNA is not possible. For instance, by addingnon-essential DNA between the two ITR sequences, the size of thereplicated DNA exceeds the packaging limit of AAV.

A cell line based on tetracycline and adenovirus regulatedrep-expression and SV40 large T-antigen based replication and expressionof cap can be generated using the following general protocol.Transfection of CV1 cells or HeLa cells can be performed with aconstruct containing the cap-gene flanked by two SV40 origins ofreplication together with a plasmid containing a dominant selectablemarker gene. Following selection, individual clones can be split intotwo fractions. one half of the cells can be transfected with anexpression construct for large T-antigen and screened for largeT-antigen dependent replication and expression of the cap-gene. Theother half of the cell lines that do express the cap-gene upontransfection, can be used to stably transfect the ptetp5repcapconstruct, or suitable derivatives with deletions in cap, and a tetp5regulated SV40 large T-antigen construct. Preferably, these constructsare transfected together but without a construct containing a dominantselectable marker gene. In co-transfections, constructs usuallyintegrate together in the same chromosomal position; thus an enhancer inthe promoter driving the dominant selectable marker gene can stimulatethe co-transfected inducible promoters. Following transfection, thecells can be seeded single cell per well in separate wells of a 96 welldish. After two weeks, the colonies can be split in two, one half of acolony can be reseeded into fresh 96 well plates and one half of thecells can be used to screen by PCR for the presence of rep and largeT-antigen DNA. Colonies containing both DNA sequences can be expandedand analyzed for the tetracycline and adenovirus regulated expression ofRep78, Rep68, Rep52, Rep40, large T-antigen and small T-antigen onWestern blots. Cells correctly expressing these proteins can beidentified upon infection with adenovirus and transfection with aconstruct pUHD15-1⁵² containing an expression cassette of the artificialtransactivator tet^(R)-Vp16. Functional expression of rep, cap and largeT-antigen can be verified by transfecting the cells with pUHD15-1,pIG-CFT and infecting the cells with adenovirus. After two or more days,rAAV produced can be analyzed with a replication center assay specificfor pIG-CFT. Cell lines that produce rAAV in such a way can be turnedinto a general packaging line for rAAV by stably transfecting thetTA-gene. The cells can be transfected with pUHD15-1 together with adominant selectable marker gene such as neo^(R). This transfection andsubsequent selection and culture should be performed in the presence oftetracycline or suitable analog to avoid inadvertent activation of thetetp5 promoters. G418 resistant colonies can be analyzed for rAAVproduction by transfection with pIG-CFT, removing the tetracycline andinfecting the cells with adenovirus. The rAAV produced can be quantifiedby RCA. Regulated expression of rep, cap and Large-T-antigen will beverified on Western blot.

Materials and Methods

Constructs: ptetp5repcap was made by cloning a 4.3 kb XbaI fragmentderived from pAAV/Ad⁷ (a kind gift from Dr. R. J. Samulski) containingthe entire rep and cap coding domain in the XbaI site immediatelydownstream of the tet(o) (FIG. 2). In ptetp5repcap, the tet(o) issituated upstream of a −68 bp p5-promoter (measured from the first baseof the TATA-Box). To completely substitute the p5-promoter for thetet(o) we performed a PCR reaction. The upstream primer was5′-ATTAATCTAGACTAGTCGCGCAGCCGCCATGCCGGGG-3′ (SEQ ID NO:1) and thedownstream primer was 5′-TGTGGAAGTAGCTCTCTCCC-3′ (SEQ ID NO 2). The PCRreaction was performed on pAAV/Ad with pfU™ (Stratagene) using thebuffer and the reaction conditions recommended by the manufacturer. Thefinal construct, ptetrepcap, was generated by digesting the 299 bp PCRproduct with XbaI and SfiI and ligating the resulting 248 bp fragment ina three part ligation with a 3.95 kb SfiI-XbaI fragment from pAAV/Adinto the XbaI site immediately downstream of the tet(o) construct. Theamplified part of the construct was sequenced and found to be asanticipated.

The ptetcap construct was derived from construct ptetrepcap by deletionof a large part of the rep-gene and the p40 promoter (self-ligation of a6.9 kb blunted SpeI and HindIII fragment). ptetp4ocap was derived fromconstruct ptetrepcap by deletion of a large part of the rep-gene butleaving a 220 bp p40 promoter fragment (self-ligation of a 7.2 kbblunted SpeI and BstB1 fragment). pMLPicap was made by ligating thecap-gene (HindIII-BamHI fragment from ptetrepcap) into the HindIII-BamHIsite of pMLPiTK. pITRp5p40cap was generated by deleting the SfiI-BstBIfragment of the rep-gene from pSM620 (a molecular clone of AAV inpBR322). ptetrep was generated as follows. The 3′ part of the rep-genewas amplified with two specific primers which also carried additionalsequences for the restriction enzymes ClaI and AvrII (upstream primer)and BglII and SpeI (downstream primer). The sequence of the upstreamprimer was 5′-GGTATCGATCCTAGGCGTCAGACGCGGAAGCTTCG-3′ (SEQ ID NO 3) andthe sequence of the downstream primer was5′-CCAACTAGTAGATCTGCTTCCACCACTGTCTTATTC-3′ (SEQ ID NO 4). The PCRreaction was performed on pAAV/Ad with pfu™0 (Stratagene) using thebuffer and the reaction conditions recommended by the manufacturer. Theamplified product was digested with ClaI and SpeI and cloned into theClaI, SpeI site of pBluescript SK⁺ to form pBluePCRREP1. The constructwas verified by sequencing and found to be as anticipated. The 1.9 kbSpeI-SwaI fragment from ptetrepcap was ligated into the AvrII-SwaI sitesfrom pBluePCRREP1 to form pBlueREP1. pBluetetREP was generated byinserting the ClaI-HindIII fragment from ptetrepcap into theClaI-HindIII sites of pBlueREP1. ptetrep was generated by ligating theBamHI-BglII SV40 polyadenylation site fragment from pMLPiTK into theBglII site of pBlueREP1.

HtTA cells⁵², 293 cells⁵⁶ and CV1 cells⁶⁰ were maintained in Dulbecco'smodified Eagles Medium (DMEM, Life Technologies, Breda, The Netherlands)supplemented by 10% heat inactivated Fetal Bovine Serum (GIBCO, LifeTechnologies, Breda, The Netherlands, FCS) and 50 ug/ml Gentamycin(GIBCO, Life Technologies, Breda, The Netherlands). The cells werecultured in 10% CO2, 100% humidity at 37° C.

Monoclonal Antibodies: The MOAB 7B7.32 specific for respectively. Rep78,Rep68 was a kind gift of R. Samulski. The MoAB B1 specific for thecapsid proteins VP1, VP2 and VP3⁶¹ was a kind gift of J. A.Kleinschmidt.

Protein extraction was performed as described in Kyostio et al⁶².SDS-PAGE gel electrophoresis and Western blotting were performed asdescribed in⁶³. MOAB detection of AAV proteins was performed asdescribed below. Nitrocellulose filters were incubated overnight (4° C.)in 5% low fat milk powder. The next day, the filters were washed withwash buffer (PBS containing 0.05% (v/v) TWEEN20). Subsequently, thefilters were incubated (1.5 hrs, room temperature) with an appropriatedilution of the monoclonal antibody (MoAB) in wash buffer supplementedwith 0.1% (w/v) BSA. The filters were washed 3 times with wash buffer.Rep and Cap proteins were visualized by peroxidase-coupled secondaryantibodies and enhanced chemiluminescence detection (ECL kit; AmershamInternational, Den Bosch, The Netherlands) as described by the supplier.

Hirt-extractions and replication center assays and crude rAAV-isolationswere performed as described in³¹. Standard molecular biology techniques,such as Northern blot analysis, cloning and filter hybridization wereperformed as described in⁶³.

Transfection of cell lines. DNA transfections were carried out usingCaPO4 precipitation kit (Life Technologies, Breda, The Netherlands)according to the manufacturer's specifications.

For the analysis of transient transfection, the cells were harvestedafter 48 hours. For stable transfections, the cells were co-transfectedwith the plasmid of interest and plasmid pX343, a hygromycin constructexpressing the hygromycin phosphotransferase (hph) gene for resistanceto hygromycin B⁶⁴. Stable cell pools carrying the plasmid of interestwere generated by selecting the cells for 10-14 days in normal mediumsupplemented with 400 ug/ml Hygromycin B (Calbiochem, La Jolla,California, U.S.A), with or without tetracycline (2 ug/ml, Sigma, StLouis, U.S.A.) or doxycycline (1 ug/ml, Sigma, St Louis, U.S.A.). Thisselection medium was replaced twice a week.

Constructs

Construction of Rep-Expression Cassettes

To obtain the final construct ptetp5repEcoNI, the following strategy wasemployed. First, the TATA-box from plasmid pUHD10-3 was removed to giveplasmid pUHD(10-3)ΔKpnI. To this end, the plasmid pUHD 10-3 of 3.15 kb(a kind gift from prof. H Bujard), containing the eptamerizedtet-operators upstream of the minimal hCMV promoter (52), was digestedwith KpnI and self-ligated. Plasmid ptetMCS (3.0 kb) was made byintroducing a multiple cloning site (MCS) into XbaI-KpnI digestedpUHD(10-3)ΔKpnI. This was done by inserting a phosphorylated doublestranded oligo carrying the restriction sites SfiI, BglI, XbaI, EcoNIand AflII with SpeI and KpnI overhanging 5′ and 3′ ends, respectively.The linker was obtained by hybridizing a sense oligo with sequence5′-CGGCCGCCTCGGCCCTCTAGAGCCTTCTTAAGGCGA-3′ (SEQ ID NO 5) to theanti-sense oligo 5′-CTAGTCGCCTTAAGAAGGCTCTAGAGGGCCGAGGCGGCCGGTAC-3′ (SEQID NO 6). ptetp5repEcoNI (5.4 kb) was then generated by inserting a 2.4kb XbaI-EcoNI fragment derived from pSub201 (7) containing the entireRep gene into XbaI, EcoNI digested ptetMCS.

We next generated a rep-expression plasmid, ptet*p5repEcoNI, with analtered −60 YY1 and Rep-binding site. Two PCR reactions were carried outresulting in a 100 bp p5 promoter fragment and a 630 bp fragmentcontaining the upstream part of the rep. Into these fragments, thespecific mutations were inserted through the primers. The 100 bpfragment was amplified with two specific primers, which also includedadditional sequences for the restriction enzyme sites KpnI, XbaI andSfiI (upstream primer) and KpnI, SpeI and SphI (downstream primer).Thesequences of the upstream and downstream primers were respectively:5′-GGGGTACCTCTAGAGTCCTGTATTAGAGGTCACGTG-3′ (SEQ ID NO 7) (pr1) and5′-CCGGTACCACTAGTACGCATGCTTAAATACCCAGCGTGACCAC-3′ (SEQ ID NO 8) (pr2).The reaction was performed on ptetp5repEcoNI with the Expand LongTemplate PCR System (GIBCO BRL) using buffer and conditions recommendedby the manufacturer. The PCR reaction to amplify the upstream part ofthe rep-gene was carried out by using the upstream primer5′-GGGGTACCGCATGCGTACTAGTCGAGGGTCTCCATTTTGAAGCGG-3′ (SEQ ID NO 9) (pr3)that also carried additional restriction enzyme sites for KpnI, SphI andSpeI and the rep-specific downstream primer 5′-AACCGTTTACGCTCCGTGAG-3′(SEQ ID NO 10) (pr4). The reaction was performed on ptetp5repEcoNI withpfu DNA polymerase (Stratagene), according to the conditions recommendedby the manufacturer. The PCR products contained a 40 bp overlap and werelinked by annealing PCR using pfu DNA polymerase, according to themanufacturer's protocol. Approximately 50 ng of each PCR product wasused as template for the annealing PCR reaction using primers pr1 andpr4. The resulting 730 bp amplified product was digested with XbaI andNcoI and cloned into XbaI-NcoI digested ptetp5repEcoNI to giveptet*p5repEcoNI. The cloning junctions and the parts derived from PCRfrom plasmids ptetp5repEcoNI and ptet*p5repEcoNI, were sequenced andfound to be as expected.

Construction of Enlarged Cap-Expression Cassettes

The replicated cap-gene in construct pITRp5p40cap is 3.5 kb and can bepackaged into AAV-capsids. To avoid this, we enlarged the replicatedfragment such that it would be too large to be encapsidated. Due to theabsence of suitable restriction sites, we had to redesign the ITRcapconstruct. An additional objective was to remove the p5-promoter fromthe ITRcap construct to avoid unnecessary homology between the rep- andthe cap-expression cassettes. The resulting construct pITR6.5capcontains between ITRs consecutively, a 538 bp fragment of the human ADAcDNA sequences, p40cap-sequences (nucl. 1624 until 4491 from AAV-2,accession number M12469 Genbank), and sequences 20901-23930 from humanADA intron2 (accession number M13792, Genbank). First, we cloned aPCR-fragment from the human ADA cDNA into NotI-site of pBluescript SK+to give pBLada. The sequence of the upstream and downstream primer wasrespectively 5′-ATAAGAATGCGGCCGCTCGCCCTCCCAGCTAACACA-3′ (SEQ ID NO 11)and 5′-AGTTTAGCGGCCGCAGATCTTCGTTCGAAGGCCTGGACATGTCCAGGC-3′ (SEQ ID NO12). The PCR reaction was performed on pAMGI (EMBO J. Vol. 4, pp 437,1985) with EXPAND long template PCR system (GIBCO BRL), according to theconditions recommended by the manufacturer. The amplified product (538bp) was digested with NotI and cloned into the NotI site of pBluescriptSK+ to form pBLada. To clone the cap-gene in pBLada, we first introduceda phophorylated double stranded NotI-BglII linker with NcoI sitenucleotide overhang into the NcoI site, 3′ of the cap-coding region inpITRp5p40cap to give pITRp5p40cap+linker. The sequences of the strandand anti-strand oligos were: 5′-CATGGCGGCCGCAGATCTC-3′ (SEQ ID NO 13)and 5′-CATGGAGATCTGCGGCCGC-3′ (SEQ ID NO 14). To introduce the p40capsequences in pBLada, we performed a three part ligation. pBLadacap wasgenerated by ligating a 780 bp BstBI-SstII cap fragment from pSM620 (amolecular clone of AAV in pBR322, kind gift from K. I. Berns) and a 2.0kb SstII-BglII fragment from pITRp5p40cap+linker into BstBI-BglIIdigested pBLada. Next, we generated an ITR-construct containing the 3.0kb fragment derived from the second intron of human ADA. The 3.0 kbfragment was derived by PCR from HtTA genomic DNA using the upstreamprimer 5′-GGACAGATCTGCGGCCGCACTCCTTTAAGTGCGTTACC-3′ (SEQ ID NO 15) andthe downstream primer 5′-GGAACAGATCTGCGATTCTCCTAATGGTCTCC-3′ (SEQ ID NO16). For cloning purposes, a BglII and a NotI site were introduced inthe upstream primer, whereas in the downstream primer, a BglII site wasintroduced. The PCR reaction was performed with expand long template PCRsystem (GIBCO BRL) using buffer and reaction conditions recommended bythe manufacturer. The PCR fragment was digested with BglII and ligatedinto BglII digested pTR+ plasmid to yield pTRintronADA (described inProc Natl Acad Sci U.S.A. Vol. 94, pp 6916-6921, 1997). Next, the finalconstruct pITR6.5cap was generated by ligating a 3.2 kb NotI-fragmentfrom pBLadacap into NotI digested pTRintronADA. Both orientations of theadacap-fragment into pTRintronADA were obtained. In pITR6.5cap, thedirection of transcription of the cap-gene is toward the ADAintron-sequence.

The pITR6.5capZEO construct was generated by ligating the 1.2 kbClaI-XbaI CMV-ZEO fragment from pZEOSV2 into ClaI-NheI digestedpITR6.5cap.

The test recombinant AAV plasmid pTR-Luc was generated by amplifying a 3kb fragment containing the CMV-Luciferase expression cassette from IG-AdCMV Luc template, described in patent application EP 95202213, withprimers 5′-CACAGATCTGCGGCCGCCAGGGGCTGCAGGTCGTTAC-3′ (SEQ ID NO 17) and5′-TGGAGATCTGCGGCCGCCCGCCACACTCGCAGGGTCTG-3′ (SEQ ID NO 18) using expandlong template PCR system (GIBCO, Life Technologies, Breda, TheNetherlands), according to the specifications of the manufacturer.Introduced into the primers were BglII-sites with which the fragment wascloned into the BglII site of pTR+.

Miscellaneous plasmids: pPur and pTK-hyg were obtained from Clontech,Palo Alto, CA USA. pZeoSV2 was obtained from Invitrogen, San Diego, CAUSA.

Probes: The Cap-specific probe is a 1,3 bp SstII-NcoI fragment fromptetp40cap. The Luciferase specific probe is a 2 kb BglII-HindIIIfragment from pTR-Luc. The ADA-specific probe is a 500 bp NotI-fragmentfrom pBLada. The rep-specific probe is a 650 bp SstI-fragment fromptetp5repEcoNI.

Monoclonal antibodies: MoAB-B1 (αVP) specific for the capsid proteinsVP1, VP2 and VP3 (Progen, Heidelberg, Germany). MoAB 303.9 specific forthe Rep proteins Rep 78, Rep 68, Rep 52 and Rep 40 (Progen, Heidelberg,Germany).

Mass Screening of Rep-expressing clones using a protein Dot blot assay.Samples to be analyzed by dot blot assay were obtained by lysing thecells from a 6 well dish in 200 microliter protein lysisbuffer (62).Samples were sonicated on ice for 15 seconds on setting 5 (SONIPREP™ 150(MSE), Beun de Ronde B. V. Abcoude, The Netherlands). Protein contentwas quantified using the Dc protein assay kit from Biorad (Veenendaal,The Netherlands). 30 and 3 microgram of protein was diluted inblotbuffer (63) devoid of SDS and spotted on IMMUNOBILONP™ transfermembranes (Millipore Corporation, Bedford, Massachusetts, U.S.A.), usinga BIO-DOT™ Microfiltration Apparatus from Biorad (Veenendaal, TheNetherlands), according to the specifications of the manufacturer. Afterspotting, the filters were incubated and processed as normal Westernblot filters.

Induction of Rep-expression in stable cell lines. To inducerep-expression in cells growing in the presence of doxycycline (1microgram/ml), the doxycycline needs to be washed away extensively. Tothis end, cells were washed by replacing the medium four times withfresh culture medium without doxycycline. Subsequently, the cells wereinfected with adenovirus ts149 at an moi of 20. Unless otherwise statedbefore, to harvest the cells, the cells were incubated for 24 hours forHirt-extraction, for 48 hours for protein extraction and for 72 hoursfor rAAV-isolation.

Titration of pTR-Luc recombinant AAV. Recombinant AAV was produced bytransfection (CaPO4 transfection kit, Life Technologies, Breda, TheNetherlands) of pTR-Luc on washed and adenovirus infected CARE.1 cellsor B1 cells according to the specifications of the manufacturer. Afterthree days, rAAV preparations were made according to the protocoldescribed in (31). To test for the presence of recombinant AAVcontaining the Luciferase gene, virus was titrated on HtTA cells oradenovirus infected HtTA cells. After 24 hours, the cells were harvestedand analyzed for Luciferase activity using the reporter lysis buffer andLuciferase detection kit from Promega (Leiden, The Netherlands) usingthe protocols supplied by the manufacturer. Detection of Luciferase wasperformed in a LUMATLB9507™ luminometer (EG and G, Berthold AG,Germany).

Example 7

Generation of a General Packaging Cell Line for rAAV-Vectors

To generate a general packaging cell line for rAAV-vectors, we firstgenerated a new series of rep-expression constructs. One objective wasto reduce the amount of cap-specific sequences from the cassette tominimize the possibility of recombination between rep and cap expressionconstructs that could lead to the formation of replication competentAAV. The other objective was to dissect the promoter drivingrep-expressior to further delineate and optimize the cis-acting elementsresponsible for inducible expression of rep.

Construct ptetp5repEcoNI was different from ptetp5repcap in that weremoved the TATA-box originating from the tet-operon and in thatsequences 3′ of the EcoNI site were deleted to minimize overlap withcap-constructs while retaining all information required for optimalsplicing of the rep-specific mRNA (see materials and methods fordetails). Construct ptet*p5repEcoNI differs from ptetp5repEcoNI in thatthe rep-binding element in the p5-promoter was replaced by a syntheticlinker and that the YY1 site at −60 was deleted (measured from thetranscription start site in the p5-promoter).

Integrity of the constructs was analyzed in transient expression inadenovirus infected HtTA cells. Protein was harvested after 48 hours andWestern blotted (FIG. 13). All constructs expressed rep. In constructptetrep, only rep-proteins derived from the unspliced messengers areclearly detected, Rep78 and Rep52. In construct ptetp5repEcoNI andptet*p5repEcoNI, products from the spliced messengers, Rep68 and Rep40,are also detected. Strikingly, in cells transfected with constructptetp5repEcoNI, Rep52 is by far the most abundant protein. Thesignificance of this is as yet not known. As discussed later, in stabletransfected cells this striking abundance of Rep52 was not observed.

To verify whether the rep was functional, we transfected the constructsinto adenovirus infected HtTA-cap2 cells and two days later harvestedrAAV. HtTA-cap2 cells contain a rescuable 3.5 kb rAAV containing thecapsid gene. Functional expression of Rep in these cells leads torescue, replication and packaging of the cap-construct (described inexample 4). For detection of the production of rAAV, the viruspreparation was infected on adenovirus infected and ptetrep transfectedHtTA cells. After 24 hours, cells were harvested and extrachromosomalDNA was isolated, Southern blotted and probed with a cap-specific probe.In spite off differences in the ratios with which the differentRep-proteins are expressed, all rep-constructs were able to complementthe cap-construct in HtTA-cap2 cells (FIG. 14).

Next, we set out to generate cell lines stably transformed with therep-expression cassette. The constructs were transfected into HtTA cellstogether with pTK-Hyg. After two weeks of selection with Hygromycin B inthe presence of doxycycline, resistant colonies became apparent. Perconstruct, between 30 and 180 colonies were picked and propagated in 96well dishes in the absence of selection but in the presence ofdoxycycline. Confluent wells were trypsinized and 10% was seeded in afresh 96 well dish and incubated at 32 degrees Celsius to slow downgrowth. The remainder was seeded in 6 well dishes to test forrep-expression. When the cells in the 6 well dishes reached a confluenceof approximately 70%, rep-expression was induced by washing away thedoxycycline and the addition of adenovirus. After 48 hours, protein wasextracted from the cells and analyzed for Rep-expression.

To get an indication for the presence of clones with the capacity toexpress Rep-proteins, we pooled the proteins from 6 clones derived fromthe transfection with construct ptetrep and from the transfection withptetp5repEcoNI. As expected, with the tet-operon construct ptetrepconstruct, no Rep-expression was detectable, whereas in the pool derivedfrom the tet-operon+p5 promoter construct, ptetp5repEcoNI,rep-expression was readily detectable (FIG. 15). To mass screen all thecolonies obtained from the transfection, we performed a Western dot blotassay in which a dilution titration of 30 and 3 microgram of protein ofeach clone was spotted on a filter. Rep-proteins were visualized withthe antibody 303.9 detecting all four Rep-proteins. An example of thisassay is given in FIG. 16. The arrows indicate protein samples stainingwith the antibody also in the lowest concentration of protein. Thesesamples were further analyzed on a normal Western blot. Clones whichgave the highest Rep-expression were expanded and stored for futurereference. One ptetp5repcap and one ptetp5repEcoNI derived clone,designated HtTA-rep1 and HtTA-rep2 respectively, were studied in moredetail. Neither the large number of clones arising with the differentclones nor the subsequent unaltered growth speed of the colonies and thetwo selected clones indicated a large toxic or growth inhibiting effectof carrying the rep-expression construct. Thus, we expected the clonesto be, at least, fairly stable and would not lose the rep-expressioncassette upon continued culture. To verify this assumption, we culturedthe clones for 26 passages without selection, but in the presence ofdoxycycline, and analyzed them first for rep-expression on Western blot.To induce rep-expression, the cells were washed extensively to removethe doxycycline and infected with adenovirus. After 48 hours, proteinwas extracted and Western blotted. Both HtTA-rep1 and HtTA-rep2expressed rep after induction (FIG. 17). Interestingly, HtTA-rep1expresses all four Rep-proteins, whereas in HtTA-rep2, Rep78 is notdetectable. To verify whether the rep-gene in the cells was functional,we analyzed whether recombinant AAV constructs were able to replicate inthese clones. To this end, the cells were washed to remove thedoxycycline and infected with adenovirus. Simultaneously, the cells weretransfected with a plasmid containing the pTR-Luc vector (FIG. 18).After 24 hours, the cells were harvested and extra-chromosomal DNA wasisolated, digested with DpnI, Southern blotted and probed with aLuciferase specific probe. Clearly detectable are DpnI resistantextra-chromosomal bands corresponding in size to the duplex monomer andthe duplex dimer of the replicated pTR-Luc vector. From theseexperiments, we conclude that the expression cassettes in HtTA-rep1 andthe HtTA-rep2 are functional. Considering the fact that the cells wereonly selected with Hygromycin B for two weeks following transfection andwere cultured and expanded without selection for more then 3 months, weconclude that the clones are stable and that there is no strongselection against the presence of the rep-expression cassette in theseclones.

In HtTA-rep1, also, the cap-gene is stabily integrated into the hostcell genome. Although Western blot analysis revealed expression of allRep-proteins, capsid gene expression was often undetectable and, in thebest experiments, very low (not shown). To generate a general packagingcell line expressing both the rep- and the cap-gene, we introduced intoHtTA-rep1 and HtTA-rep2 cells a construct, pITR6.5cap, in which thecap-gene is flanked by two AAV-TR. To prevent packaging of thisrAAV-molecule, the total size of the rAAV was enlarged to 6.5 kb byintroducing sequences from the human Adenosine Deaminase (hADA) gene.HtTA-rep1 and HtTA-rep2 cells were co-transfected with constructpITR6.5cap and pPur, a plasmid expressing the dominant selectable markergene puromycin. After transfection and a two week selection in puromycin(2.5 microgram/ml), individual puromycin resistant colonies were pickedand expanded in the absence of puromycin. To select clones stabilytransformed with at least one intact copy of the AAV-cap construct,cells from individual colonies were seeded in 6 well dishes in theabsence of doxycycline and infected with adenovirus. After 36 hours, thecells were harvested and extrachromosomal DNA was isolated, Southernblotted and probed with a hADA specific probe. One clone out of the 14colonies tested, derived from the transfection of HtTA-rep1 cells,produced a detectable amount of excised and extrachromosomaly replicatedAAV-cap construct. This clone, designated CARE.1, and a negative clone,B1, were expanded and the replication experiment was repeated (FIG. 19,panel A). As expected, no replicated cap-expression cassette wasdetected in uninduced CARE.1 cells. Upon induction with adenovirus inthe absence of doxycycline, excised and replicated cap-expressioncassettes are readily detectable, even on a 10 minute exposure of theSouthern blot (FIG. 19, panel A). Neither the negative clone B1, nor theparent HtTA-rep1 or the HtTA cell line contain this fragment. To excludethe possible generation of replication competent AAV, the Hirt-extractswere analyzed with a rep-specific probe (FIG. 19, Panel B). No specificbands of approximately 4.7 kb are detected, even after overnightexposure. There is some signal in the high molecular weight range thatcorresponds most likely to non-specific hybridization to residualchromosomal and adenovirus DNA. In contrast to the safe integratedstate, we detected in the transient transfection of pITR6.5cap andptet*p5repEcoNI on adenovirus infected HtTA cells a replicationcompetent rAAV carrying rep-gene sequences. The size of this fragment is3.5 kb for the duplex monomer and approximately 7 kb for the duplexdimer, which is too small for both the rep- and the cap-gene to bepresent intact on this recombination product. We next tested theexpression of rep- and cap-gene products in CARE.1 cells. UninducedCARE.1 cells do not express rep or cap-gene products (FIG. 20). Uponinfection with adenovirus in the absence of doxycycline, Rep78, Rep68and a small amount of Rep52 are detected. Readily detected are also VP1,VP2 and VP3. Moreover, the relative ratio of the three capsid proteinsmimics the relative abundance of the proteins in AAV-capsid and in wtAAVproducing cells. As expected, the clone B1, from which no pITR6.Scap isrescued and replicated, expresses upon induction only rep-gene products.The also present cap-expression cassette is not expressed in clone B1,also not upon adenovirus infection. The ptetp5repcap expression cassetteis not rescued and replicated by the AAV-rep proteins produced. NormalHtTA cells served as a negative control and, as expected, do not expresseither Rep or Cap.

Next, we determined virus production by the CARE.1 cells. CARE.1 cellsand, as a negative control, B1 cells were washed, infected withadenovirus and transfected with pTR-Luc. After three days, virus washarvested and used to infect normal HtTA cells and adenovirus infectedHtTA cells. After 24 hours, Luciferase was measured. In contrast to rAAVproduced on B1 cells, rAAV produced on CARE.1 cells produced detectableluciferase activity in target cells and, thus, produced rAAV (Table 1).

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TABLE 1 Luciferase activity in rAAV infected HtTA rAAV rAAV Dilutionfrom from of rAAV clone B1 CARE.1 −Ad +Ad −Ad +Ad 2 100 51 2269 2656 50294 37  226  502

18 1 37 DNA Artificial Sequence Description of Artificial SequenceUpstream primer 1 attaatctag actagtcgcg cagccgccat gccgggg 37 2 20 DNAArtificial Sequence Description of Artificial Sequence Downstream primer2 tgtggaagta gctctctccc 20 3 35 DNA Artificial Sequence Description ofArtificial Sequence Upstream primer with additional sequence forrestriction enzymes ClaI and AvrI 3 ggtatcgatc ctaggcgtca gacgcggaagcttcg 35 4 36 DNA Artificial Sequence Description of Artificial SequenceDownstream primer with additional sequence for restriction enzymes BglIIand SpeI 4 ccaactagta gatctgcttc caccactgtc ttattc 36 5 36 DNAArtificial Sequence Description of Artificial Sequence Sequence is senseoligo and is hybrizied to anti-sense oligo to create linker 5 cggccgcctcggccctctag agccttctta aggcga 36 6 44 DNA Artificial Sequence Descriptionof Artificial Sequence Anti-sense oligo portion of linker 6 ctagtcgccttaagaaggct ctagagggcc gaggcggccg gtac 44 7 36 DNA Artificial SequenceDescription of Artificial Sequence Upstream primer with sequences forrestriction enzymes KpnI, XbaI and SfiI 7 ggggtacctc tagagtcctgtattagaggt cacgtg 36 8 43 DNA Artificial Sequence Description ofArtificial Sequence Downstream primer with sequences for restrictionenzymes KpnI, SpeI and SphI 8 ccggtaccac tagtacgcat gcttaaatacccagcgtgac cac 43 9 45 DNA Artificial Sequence Description of ArtificialSequence Upstream primer with sequences for restriction enzymes KpnI,SphI and SpeI that is used to carry out rep-gene amplification 9ggggtaccgc atgcgtacta gtcgagggtc tccattttga agcgg 45 10 20 DNAArtificial Sequence Description of Artificial Sequence Rep-specificdownstream primer 10 aaccgtttac gctccgtgag 20 11 36 DNA ArtificialSequence Description of Artificial Sequence Upstream primer 11ataagaatgc ggccgctcgc cctcccagct aacaca 36 12 48 DNA Artificial SequenceDescription of Artificial Sequence Downstream primer 12 agtttagcggccgcagatct tcgttcgaag gcctggacat gtccaggc 48 13 19 DNA ArtificialSequence Description of Artificial Sequence strand oligo 13 catggcggccgcagatctc 19 14 19 DNA Artificial Sequence Description of ArtificialSequence Anti-strand oligo 14 catggagatc tgcggccgc 19 15 38 DNAArtificial Sequence Description of Artificial Sequence Upstream primerwith sequences for restriction enzymes BglII and NotI 15 ggacagatctgcggccgcac tcctttaagt gcgttacc 38 16 32 DNA Artificial SequenceDescription of Artificial Sequence Downstream primer with a sequence forrestriction enzyme BglII 16 ggaacagatc tgcgattctc ctaatggtct cc 32 17 37DNA Artificial Sequence Description of Artificial Sequence Primer 17cacagatctg cggccgccag gggctgcagg tcgttac 37 18 38 DNA ArtificialSequence Description of Artificial Sequence Primer with BglII site 18tggagatctg cggccgcccg ccacactcgc agggtctg 38

What is claimed is:
 1. A method for producing a nucleic acid which can be conditionally replicated, said nucleic acid not comprising a rep gene, but comprising at least a functional part of a cap gene of Adeno Associated Virus (AAV), which nucleic acid is present integrated in the genome of a eukaryotic host cell, said method comprising providing said nucleic acid with at least one regulatory element which essentially represses replication of said nucleic acid in an integrated setting, but allows replication in an episomal setting, and further providing said nucleic acid with at least a means for functionally excising said nucleic acid in a functional form upon the presence of a signal for excision, thereby producing a nucleic acid which can be conditionally replicated, wherein replication is conditional on said nucleic acid being in an episomal functional form.
 2. The method according to claim 1 wherein a means for excision is provided in the form of two AAV-ITR's on either side of the nucleic acid of interest.
 3. The method according to claim 1 wherein the signal is at least a functional part of a Rep-protein of AAV.
 4. The method according to claim 1 wherein said means for excision is from a viral replication system.
 5. The method according to claim 4 wherein the excision means is from a papova virus.
 6. The method according to claim 5 wherein the signal is at least a functional part of the large T-antigen.
 7. A method for producing a nucleic acid which can be conditionally transcribed which nucleic acid is at least a part of a DNA molecule of interest which DNA molecule does not comprise a rep gene, but comprises at least a functional part of a cap gene of Adeno Associated Virus (AAV) and is present integrated in the genome of a eukaryotic host cell, said method comprising providing said DNA molecule with at least one regulatory element which essentially represses replication of said DNA molecule in an integrated setting, but allows replication in an episomal setting, further providing said DNA molecule with at least a means for functionally excising said DNA molecule in a functional form upon the presence of a signal for excision, thereby producing a nucleic acid which can be conditionaly transcripted, wherein transcription is conditional on said DNA molecule being in an episomal functional form.
 8. The method according to claim 7 wherein transcription is under control of an inducible promoter.
 9. The method according to claim 7 wherein a means for excision is from a viral replication system.
 10. The method according to claim 7 wherein means for excision is provided in the form of two AAV-ITR's on either side of the DNA molecle of interest.
 11. The method according to claim 7 wherein the signal is at least a functional part of a Rep-protein of AAV.
 12. A method for producing a nucleic acid molecule of interest which can be conditionally expressed which nucleic acid molecule of interest does not comprise a rep gene, but comprises at least a functional part of a cap gene of Adeno Associated Virus (AAV) and is present in the genome of a eukaryotic host cell, said method comprising providing said nucleic acid molecule of interest with at least one regulatory element which essentially represses replication of said nucleic acid of interest in an integrated setting, but allows replication in an episomal setting, further providing said nucleic acid of interest with at least a means for functionally excising said nucleic acid of interest in a functional form upon the presence of a signal for excision, thereby producing a nucleic acid molecule of interest which can be conditionally expressed, wherein expression is conditional on said nucleic acid of interest being in an episomal functional form.
 13. The method according to claim 12 wherein transcription is under control of an inducible promoter.
 14. The method according to claim 12 wherein means for excision is provided in the form of two AAV-ITR's on either side of the nucleic acid of interest.
 15. The method according to claim 12 wherein the signal is at least a functional part of a Rep-protein of AAV.
 16. A recombinant vector for carrying out a method for producing a nucleic acid molecule which can be conditionally expressed which nucleic acid molecule does not comprise a rep gene, but comprises at least a functional part of a cap gene of Adeno Associated Virus (AAV) and is present integrated in the genome of a eukaryotic host cell, said recombinant vector comprising a nucleic acid sequence of interest, means for integration into the genome of the eukaryotic host cell, means for functional excision of the nucleic acid sequence of interest after integration upon the presence of a signal for excision, means for essentially repressing replication of said nucleic acid sequence of interest in an integrated setting, and means for allowing replication of the nucleic acid of interest in an episomal setting, wherein one or more of the mentioned means may be one and the same.
 17. The recombinant vector of claim 16 further comprising an inducible promoter.
 18. The recombinant vector of claim 16 wherein at least one of the mentioned means is from AAV.
 19. The recombinant vector of claim 18 comprising at least one of the following elements: a functional part of a cap-gene and an ITR from AAV.
 20. The recombinant vector of claim 16 wherein at least one of the elements is from a papovavirus.
 21. The recombinant vector of claim 20 comprising an SV40 origin of replication.
 22. The recombinant vector of claim 16 further comprising at least a functional part of a rep-gene.
 23. The recombinant vector of claim 22 wherein the rep-gene is under control of a combination of two inducible repressor/activator sequences.
 24. A recombinant eukaryotic host cell comprising a recombinant vector for carrying out a method for producing a nucleic acid molecule which can be conditionally expressed which nucleic acid molecule does not comprise a rep gene, but comprises at least a functional part of a cap gene of Adeno Associated Virus (AAV) and is present integrated in the genome of a eukaryotic host cell, said recombinant vector comprising a nucleic acid sequence of interest, means for integration into the genome of the eukaryotic host cell, means for functional excision of the nucleic acid sequence of interest after integration upon the presence of a signal for excision, means for essentially repressing replication of said nucleic acid sequence of interest in an integrated setting, and means for allowing replication of the nucleic acid sequence of interest in an episomal setting, wherein one or more of the aforementioned means may be one and the same.
 25. The cell of claim 24 further comprising a vector encoding at least a functional part of a rep-gene of AAV.
 26. The cell of claim 25 which comprises all AAV genes in trans, wherein said AAV genes were excised from recombinant AAV vector. 